University of Ghana http://ugspace.ug.edu.gh QP572. T4 Of2 bite C.l G374184 University of Ghana http://ugspace.ug.edu.gh VARIATIONS IN SERUM TESTOSTERONE LEVELS IN GHANAIAN MALES VIDA OFEI DEPARTMENT OF CHEM ICAL PATHOLOGY UNIVERSITY OF GHANA MEDICAL SCHOOL KORLE BU TEACHING HOSPITAL MASTER OF PHILOSOPHY (BIOMEDICAL SCIENCES CHEM ICAL PATHOLOGY) 2003 University of Ghana http://ugspace.ug.edu.gh DECLARATION THE WORK DESCRIBED IN THIS REPORT WAS CARRIED OUT BY ME AT THE DEPARTMENT OF CHEMICAL PATHOLOGY, UNIVERSITY OF GHANA MEDICAL SCHOOL, KORLE-BU UNDER THE SUPERVISION OF DR. O. A. DUAH AND DR. SAMUEL Q. MADDY Date:....3/..“ .. L !srr. J .. MW.n. Signature:...... / / Vida Ofei Date:... ..! 3 j. . Signatured.. ^T7.: Dr. O.A. Duah Supervisor Date:. Signature* Dr. S.Q. Maddy Co-Supervisor University of Ghana http://ugspace.ug.edu.gh DEDICATION To my Son Darko, His Wife Aku, Their Children Nana Ofei and Mama Opeibea. University of Ghana http://ugspace.ug.edu.gh flCXHO‘WccE 90%. 12 University of Ghana http://ugspace.ug.edu.gh Hypothalamus (SHBG), (Whitby et al., 1993). 13 University of Ghana http://ugspace.ug.edu.gh Valero-Politie et al., (1996) demonstrated that serum concentrations of SHBG and testosterone were significantly affected by 24 and 12 hrs rhythmic components. The salivary concentrations o f testosterone showed the greatest daily rhythmic variation. The serum concentration of FSH and LH showed no significant rhythmic variation daily. 14 University of Ghana http://ugspace.ug.edu.gh 2.4. Peripheral Metabolism of Plasma Testosterone Plasma Testosterone (5 mg/day) Hydroxylases Aromatase 0.3% Plasma Plasma Estradio Dihydrotestosterone (0.02 mg/day) (0.3 mg/day) 17-Ketosteroids Polar metabolites Androsterone + Diols, triols, and Active Metabolites etiocholanolone Conjugates (2.mg/day) (2.5mg/day) Excretory metabolites Fig. 23. Pathways of peripheral metabolism of plasma testosterone. Testosterone can be metabolised either to active metabolites or to excretory metabolites (Griffin et al., 1980). The liver is the principal site of degradation of testosterone and releases water soluble sulphate or glucuronide conjugates into the blood for excretion in the urine. 15 University of Ghana http://ugspace.ug.edu.gh 2.5. The Relationship between Testosterone and Other Androgens Testosterone concentrations adequately reflect androgen statuses in the adult male, since other circulating androgens, though present in comparable or even greater concentrations, are biologically weak. In healthy women the circulating testosterone level is very low, (1/10 that of men). Of this, 25% is derived from direct ovarian secretion and 25% from the adrenal cortex, but peripheral conversion of biologically weak androgens account for 50% or more o f total androgenic action in the female. These weak androgens are involved in testosterones action and production. The adrenals are the major source of dehydroepiandrosterone (DHEA) and DHEA- sulphate (DHEA-S) and to a lesser extent androstenedione. The adrenal androgens exhibit diurnal variations in output. Although DHA and DHA-S are biologically weak androgens, they serve as precursors of the more potent androgens, that is androstepedione and to a lesser extent testosterone. In healthy women the fluctuation of the testosterone level is less than the 20% seen in males. Longcope et al., (1996) found that DHEA and DHEA-S circulate in blood mostly bound to albumin, but with a small amount not bound to a protein. Dehydroepiandrosterone is cleared rapidly from the blood with a metabolic clearance rate (MCR) in the range of 2,000/day, but the clearance of DHEA-S is much slower and its MCR is in the range o f 131/day. DHEA-S will re-enter as DHEA. Both DHEA and DHEA-S can be converted in peripheral tissues to androstenedione, testosterone and dihydrotestosterone, and both are aromatised to eostrogens. DHEAS enters the ovarian follicle and can be an important source of ovarian testosterone. Most of the 17-oxosteroids in women, and to a lesser extent in men, are derived from adrenal weak androgens DHEA and androstedione. Thus urinary 17-oxosteroids therefore reflect total androgen production, but its measurement is of no use in assessing testosterone production in either sex (Rudd 1983). 16 University of Ghana http://ugspace.ug.edu.gh 2. 6. The embryonic development of the gonads and the role of testosterone. The genetic sex is determined at fertilization; the presence of a 46 XY chromosome ensures normal testicular development. In the short arm of the Y chromosone, there is a gene which is involved in the formation of a male specific cell-surface histocompatibility Y (H-Y) antigen, however there is good evidence that the H-Y antigen mediates the differentiation of the bipotential gonad into testis, (Seanger 1984, Haseltine and Oho no 1981). The primitive gonads are usually identical in both sexes; Mullerian duct in the female and Wolfian duct in the male. After seven weeks they differentiate into testes or ovaries. The Sertoli cells o f the testis are believed to produce Mullerian regression factor, or anti mullerian hormone, which appears to inhibit the further development of the female genital tract and to cause its regression. The production of testosterone stabilises the Wolffian duct, and induces its differenciation into the epididymis, vas deferens and seminal vesicles. (Imperalto Mcgingley 1983, Siiteri and Wilson, 1974). Testosterone acts directly in these tissues as 5a-reductase is not present at this time of development, (Siiteri 1974). The differentiation of the genetalia into penis, scrotum, prostate, etc., is dependent on intracellular 5a-reductase activity, to convert testosterone into 5 a Dihydrotestosterone ( 5 a DHT), which is the intracellular mediator in these tissues. Thus the differentiation of the external genetalia depends mainly on Dihydrotestosterone and not testosterone itself. 17 University of Ghana http://ugspace.ug.edu.gh The importance of androgen action in sexual development is highlighted in the human syndrome called testicular feminization, which can be traced to inherited defect in a single gene on the X chromosome. Afflicted individuals have a normal female phenotype but no menstrual cycles. Genetically they are males and have intra abdominal testis and circulating concentration of testosterone and oestrogen that are characteristic of normal men, but their tissues are unresponsive to androgens. In these individuals there is absence of androgen receptors, and because anti Mullerian hormone production and responsiveness were normal and their Wolffian ducts were unable to respond to androgens, both of these duct systems regressed and neither male nor female internal genitalia developed. Secondary sexual characteristics including breast development appear at puberty in response to unopposed action of oestrogens formed extra gonadally from testosterone. Another conversion which testosterone undergoes in some peripheral tissues as well as in testicular Leydig cells is aromatization to 170- oestradiol. The testes secrete small quantities but its role in development and maintenance o f male reproductive functions is obscure. 2. 7 Serum Testosterone Concentration from the Neonatal Period to Adulthood. Serum testosterone levels differ in neonates but are the same through childhood until adulthood is reached. In male infants there is an increase in testosterone levels on the first day of life due to decreased clearance and this may reach adult level of testestorone after separation of the placenta plus continual stimulation of residual hCG (Winter et al., 1972 and Tapanainen 1983). These levels fall within the first week and thus the testosterone concentration in both sexes then become similar (Winter, 1982) 18 University of Ghana http://ugspace.ug.edu.gh TABLE 2.1. Plasma testosterone concentrations in infancy (Winter, 1982 and Forest et al., 1976) Age Male subjects Female subjects 1 day 6-9 (2.1-19.8) 1.6 (0.7-2.7) 1 week 0.9(0.52-1.74) 0.5 (0.17-1.21) 1 - 4 months 6.2(1.74-12.5) 0.24(0.1-0.7) 4 —12 months 0.17 (0.1-.35) 0.21 (0.1-0.7) 1—2 years 0.12(0.1-0.69) 0.21 (0.1-0.7) Results (nmol/L) are expressed as median with the range in parentheses. 19 University of Ghana http://ugspace.ug.edu.gh During childhood the circulating levels of gonadal steroids and gonadotrophins are very low, but the prepubertal testis is relatively more responsive to hCG stimulation than in the adult, (Forest 1976, and Judd 1979). During childhood the testosterone concentration is low but at the onset of puberty pulsatile increases of gonadatrophins occur, at first only during the night and later during the day and night. In boys, this occurs between 12-14 years of age, (3-4 pubertal stage). In girls the increases are veiy slow throughout puberty (Table 2.2). Adult levels are usually achieved after 20 years of age. Crofton et al., (1997) demonstrated that in boys, testicular production of inhibin B increases as puberty progresses. They also show that the initiation of puberty is accompanied by a dramatic switch from a positive to negative relation between inhibin B and FSH as inhibin B begins to exert the expected negative feedback on FSH. In girls the ovarian follicles produce inhibin A & B in strict proportions and in progressively greater amounts as puberty proceeds. Thus measurement o f dimeric inhibin A& B may provide a sensitive new tool for determining gonadal maturity in late pre-puberty and early puberty. Byrd et al., (1998) also found that, inhibins, which are glycoprotein members of the transforming growth factor-beta family, have been implicated in the control of spermatogenesis by exerting a negative feedback on FSH secretion. There are inhibin A & B in serum, seminal plasma and urine. Inhibin B but not A was measurable in the serum of male newborns, infants, children and adults. The circulation levels of inhibin B increased shortly after birth and peaked at 4-12 months of age (210± 31 pg/ml) and then decreased to a low of 81± 12 pg/ml from 3-9 years and gradually increased with the onset of puberty. It reached another peak of 167± 20pg/ml in males who were 20 - 30 yrs o f age. 20 University of Ghana http://ugspace.ug.edu.gh TABLE 2.2. Plasma testosterone concentrations in relation to Stages of puberty (P1-P5) and adulthood (Ducharme et al., 1982) Age Male subjects Female subjects PI (pre-pubertal) 0.4 ± 0.24 0.4 ± 0.24 P2 0.6 ± 0.56 0.7 ±0.17 P3 1.8 ±0.87 1.0 ±0.31 P4 5.9 ±2.70 1.7 ±0.42 P5 12.1 ±5.2 1.3 ± 0.10 Adults 20 yrs. & above 19.5 ±5.5 1.6 ±0.5 Values are given as mean ± SEM (nmol/L) 21 University of Ghana http://ugspace.ug.edu.gh Rilling et al., (1996), demonstrated significant increases in plasma testosterone across puberty. Plasma testosterone distinguished among subjects at different stages o f genital development more effectively than salivary testosterone. This suggests that plasma testosterone is a better marker of testosterone bioavailability. Adult levels may not be achieved until the second decade, since a slow rise in testosterone occurs up to this time. During puberty, episodic bursts of LH and testosterone are prominent during sleep and much higher testosterone values may be found during this period. Mitumura et al., (1999) found that the diurnal rhythms of LH, FSH and testosterone already exists at 4 -5 years of age and that serum LH, FSH and testosterone levels increase before the onset of puberty. In adult males, testosterone secretions are episodic so that plasma levels are variable. There is also a circadian rhythm with the highest levels seen in early morning and the lowest in the evening, (Judd 1979). The morning levels are approximately 20-40% higher than those in the evening. This change is much smaller as compared to the other androgens whose precursors are from the adrenals, that is androstenedione and 17- hydroxy-progesterone (17-OHP), which show circadian changes which parallel those of cortisol (Vermeulen, 1996). Luteinizing hormone concentrations fluctuate in pubertal boys and men but plasma FSH levels remain more constant, (Judd. 1979). Maes et al., (1997) on the other hand found that seasonal and biannual variations in testosterone were not significant. This is followed by a gradual decline with increasing age up through 90 years o f age. 22 University of Ghana http://ugspace.ug.edu.gh Serum levels o f LH and FSH increase during the first few months of postnatal development, followed by a decrease to basal levels until the onset of puberty at 10 - 14 years of age. Testosterone also increases in infants from day 1 to 12 months and then decreases in young children but increases again following the elevation of gonadotrophins during puberty. In adults aged 20 - 90 yrs serum levels o f inhibin B was inversely proportional to levels of FSH but not LH or testosterone. As Inhibin B secretion is high in prepubertal testis up to 3yrs o f age, it could be used as a good marker in the diagnosis of cryptorchism and precocious puberty. Wang et al., (1982), found that 16 - 64mg/d, 5 a DHT gel application over both arms shoulders and upper abdomen produced an increase in testosterone. Thus they concluded that DHT gel might provide adequate androgen replacement therapy in hypogonadal men. Lawrence et al., (1997) found that men on erythropoietin (rHuEpo) therapy during the treatment o f anaemia are characterized by higher levels of serum testosterone and sex hormone binding globulin as well as increased LH and FSH. 2. 8 Factors affecting Testosterone, LH and FSH Giusti et al., (1999), demonstrated that leptin has effect on LH, FSH and testosterone secretions. This experiment was done on boys with delayed puberty. These subjects were put on gonadotrophin releasing hormone (Gn-RH) and blood samples were taken every 30 days after the start. Luteinizing hormone, FSH and testosterone increased with a decrease in leptin. Leptin increases from pre-pubertal to early pubertal stage and then declines in the late pubertal stages. 23 University of Ghana http://ugspace.ug.edu.gh Ambrosius et al., (1998) also explored the effects of race, age and sex hormones on the serum leptin concentrations in white and black children and adolescents, (ages 9-13-20 years). A significant difference in sex and race interaction on serum leptin level was observed, with lower serum leptin concentrations in black boys than in white boys. There were also differences in age by sex with serum leptin concentrations decreasing in boys than in girls. A strong inverse relationship o f serum testosterone levels with serum leptin levels were observed in boys. A higher testosterone concentration in boys appears to account for an age-related decline in serum leptin in boys and the overall lower levels in boys than in girls. Zmuda et al., (1996) also found out that there were elevations in testosterone and SHBG during exercise. These increases were transient, which is partly due to an increase in serum SHBG concentrations. The concobitant increases in total protein and the rapid return of total protein and SHBG to baseline values after exercise indicate that heamoconcentration partly contributes to the exercise associated increases in circulating testosterone levels. Fahmer et al., (1998) , also found out that exercise induced increases in testosterone, and this involved increased production which may be mediated by sympathetic stimulation o f the testicles, but had no effect on the binding affinity of SHBG. 24 University of Ghana http://ugspace.ug.edu.gh Dorgan et al., (1996) demonstrated that dietary fat and fibre has effect on testosterone. Mean plasma concentrations of total and SHBG bound testosterone were 13% and 15% higher respectively in the high-fat, low fibre diet and the difference from the low-fat, high fibre diet was significant for the SHBG bound fraction. Men’s daily urinary excretion of testosterone also was 13% higher with the high — fat, low-fibre diet than with the low-fat high-fibre diet, showing that diet might alter endogenous sex hormone metabolism in men. Tibblin et al., (1996) demonstrated that total, free testosterone and SHBG, concentrations correlated negatively with glucose and insulin values. Men newly diagnosed for diabetes, and men who were previously diagnosed as diabetics, had in general, lower total and free testosterone and SHBG levels, while values for LH were not different. These findings concluded that testosterone and SHBG concentrations in the elderly men are associated with established risk factors for diabetes and in established diabetics. Moreover, low testosterone levels independently predict the risk of developing diabetes. In different degrees of expression of the diabetic status they also predict strongly myocardial infarction and stroke.lt has been sbggested that relative hypogonadism might be a primary event, because other studies have shown that testosterone deficiency is followed by insulin resistance, which is ameliorated by testosterone substitution. Mulligan et al., (1997) demonstrated that in older men there was diminished stimulation of LH concentration on testosterone secretion rates, as well as delayed feedback inhibition of testosterone concentrations on LH secretion rates. 25 University of Ghana http://ugspace.ug.edu.gh Leifke et al., (2000) after studying the morning levels of total testosterone, free testosterone, bioavailable testosterone, oestradiol, bioavailable oestradiol, oestrone, sex hormone binding globulin and insulin-like growth factor 1 in 522 male volunteers found out that age was negatively related to serum levels of sex steroids and IGF-1, (youngest to oldest, (20yrs — 80yrs). Ratios were 51% for total testosterone, 64% for free Testosterone, 78% for bioavailable testosterone, 32% for oestradiol, 29% for oestrone and 51% for IGF-1. SHBG increased after the 5th decade of life. The decline started from the 3rd decade of life. The ageing was characterized by progressive decline in organ function associated with gradual reduction in muscle mass, bone mineral density and an increase in body fat (Lamberts et al., 1997). These signs closely resemble the well known stigmata of gonadal and of GH-deficiency of middle aged subjects, and suggested an important role of both hormonal systems in the ageing process, (Kaufman and Vermeulen, 1997, Lamberts et al., 1997, Cummings and Merriam 1999 and Meikle 1999). Data on hormone replacement therapy in the elderly indicate that hormone substitution therapy may prevent or even reverse the decline in bone mineral density and the shift from muscle to fat mass observed in ageing (Rudman et al., 1990, Corpas et al., 1993, Snyder et aL, 1999 a, b). 26 University of Ghana http://ugspace.ug.edu.gh 2. 9. Drugs Affecting Testosterone Levels. Oestrogens e.g. Diethylstilbestrol is commonly used in men in the treatment o f androgen dependent prostatic cancers; it reduced blood testosterone levels into the normal female range. SHBG increased thereby further decreasing free, testosterone. Oestrogen in oral contraceptives and during pregnancy caused up to 3-fold increase in testosterone concentrations due to total increase in SHBG (Kent et al., 1972). Danazol: used in the treatment of endometriosis, caused a 20% fell in SHBG and displaced testosterone from it. There was an increase in free testosterone up to 90% (Schwartz and Boyd 1982, Nilson et al., 1983 and Hanning, 1982). Cyproterone acetate: Is an anti-androgen used in the treatment of male hypersexuality and precocious puberty in girls and boys and sometime in hirsutism. It suppressed plasma testosterone in adult meri but not in children with precocious puberty and decreased testosterone in hirsute women (Kauli 1976, Olivo et al., 1970 and Mowszowicz etal., 1984) Garcia-Pascual et al., (1996) after the administration of desmopressin (DDAVP) 2.5 micrograms/12hours) found out that there were significant changes in the concentrations of FSH and LH after 9 days with DDAVP therapy. Serum concentrations of testosterone after 12 hours of DDAVP administration increased significantly higher than basal concentration. Three hours after the administration, the drug caused a decrease initially but possibly due to a “rebound effect” testosterone increases again. Thus they therefore concluded that, low doses of desmopressin did not change serum concentrations FSH and LH, but there was a decrease followed by an increase in testosterone; and that desmopressin would directly act on human testicles. 27 University of Ghana http://ugspace.ug.edu.gh Cytotoxic drugs: Mchlorethamine, vinblastine, procarbine and prednisone caused significant Leydig cell dysfiicntion resulting in a lower mean testosterone, higher mean LH and FSH levels, (Howell et al., 1999). Cyclophosphamide caused a decrease in testosterone due to testicular atrophy (Kumar, 1972). Anabolic steroids: 19-norandrostenololylaurate have long term effects on reproduction. Two years after treatment, testosterone levels were higher in treated patients than untreated controls. But this was not statistically significant. The treatment had no effect on libido (Koskinen and Katila, 1997). Mokshagundam and Minocha., (1997), when investigating the effect of comeprazole and ethanol on pituitary gonadal axis found out that total testosterone levels before and after ethanol at baseline, declined an average of 46.6ng/dl with a non significant probability. The testosterone level after ethanol following omeprazole therapy rose to an average of 55.4ng/dl with a non-significant probability. There was significant difference in the change of ethanol induced testosterone concentrations as a result o f omeprazole alone or in combination. Thus omeprazole and or acute ingestion of ethanol did not affect the pituitary gonadal axis in healthy male subjects, (Behre et al., 1997). Wu et al., (1999) found that oral progestogen-desogestrel (DSG) administered with low dose testosterone enanthate, were highly effective in suppressing pituitary- testicular functions in adult men. The optimal regimen for inducing azoospermia was 300mg DSG daily with 50mg testosterone enanthate weekly. Oral DSG decreased HDL cholestrol and led to the conclusion that, the combination of oral progestogen with low dose Testosterone enanthate was a promising approach to achieve effective reversible male contraception. 28 University of Ghana http://ugspace.ug.edu.gh Thyroid hormone: The increase in SHBG in thyrotoxicosis results in a 2 to 3 fold increase in total testosterone level. This was reversed in hypothyroidism, (Olivo et al., 1970, Ridgeway et al., 1975, and Ruder et al., 1971). Dopamine antagonists: Bromocriptine caused an increase o f about 20% in testosterone with no concurrent change in LH level. Dopamine antagonists such as metoclopramide generally caused decrease in testosterone although sulpride and pimozide, which increased the prolactin levels, had no effect on serum testosterone (Nakagawa et al., 1982, Carter et al, 1978, and Siris 1980). Anti psychotic drugs: Thioridazine caused slight suppression in testosterone in patients on long-term therapy. Other neuroleptic agents such as haloperidol and primozide did not alter testosterone levels, (Brown et al., 1981). Spironolactone: The anti androgenic properties were caused by displacing DHT from its cytosol receptors. It also decreased plasma testosterone levels, (Messina et al., 1983, and Spandri et al., 1984). Histamine H2 receptor antagonists: Cimetidine has anti androgenic properties, because it displaced DHT from its binding sites at hypothalamic and pituitary levels, (Funder and Mercer, 1979), and caused up to 20% increases in testosterone level in patients on chronic administration, (Wang et al., 1982). Ranitidine more powerful histamine H2 receptor antagonist had no anti- androgenic activity (Pedan et al, 1981 and Savarino et al, 1983). 29 University of Ghana http://ugspace.ug.edu.gh Anti epileptic drugs: Carbamazepine, phenytoin, primidone and sodium valproate caused reduction in free testosterone concentration. There was usually no change in total testosterone, but a small rise might occur (Barragg et al., 1978, Dani-Heari and Oxley 1982, Toone et al 1980, Toone et al 1982). Antibiotics: Tetracyclines reduced both total and free testosterone by about 10-20% but had no effect on SHBG concentrations, (Pukkinen and Maepaa., 1983). Rifampicin caused an increase in testosterone, probably due to increased in microsomal activity and increased biosynthesis of testosterone (Nocke-Finck et al., 1980). Lawrence et al., (1997), revealed that dialysing patients being treated with erythropoietin (rHuEpo) for correction of anaemia, were characterised by increased levels o f testosterone and SHBG as well as LH and FSH. Fitzgerald et al., (1997) found out that men receiving glucocorticosteroids for chronic inflammatory diseases had a reduction in free testosterone. Kadioglu et al., (1999) revealed that in patients with normogonadotrophic oligozoospermia, tamoxifen citrate might be offered as a practical and economical alternative before using any assisted reproductive technique as this was shown to increase the level of FSH, LH and testosterone. Normo-gonadotrophic patients had a significant increase in sperm count and gonadotrophin concentration. Uygur et al., (1998) when investigating the effect of finasteride a steroid 5 alpha reductase inhibitor, given orally (5 mg/day), found out that the drug had a negative effect on the serum levels of gonadal, hypophyseal, and adrenal hormones. The duration of this study was for 3, 6 months and more than 6 months, in about 75% who were judged potent before 30 University of Ghana http://ugspace.ug.edu.gh the treatment resulted in erectile dysfunction in 22% of the patients by month 3 and 33% by month 6. On the other hand there was substantial improvement in symptoms of benign prostatic hyperplasia in all patients. There were decreased prostate specific antigen level of 42% and 50% at month 3 and at month 6 respectively. After the 6th month, continued administration of the drug caused decreased testosterone, dihydrotestosterone, FSH and LH significantly. It also caused sexual dysfunction in a substantial number of patients. 2.10. Conditions Needing Testosterone Measurement. The measurement of testosterone is in the investigation of androgen-related disorders. The clinical presentation of disorders of androgen production or action varies greatly depending on age sex and ethnic grouping. At birth and during infancy, the main problems encountered are cryptorchidism and ambiguous genitalia. During childhood, the two main disorders are precocious puberty in boys and virilisation in girls. In adolescent and adult males, the main clinical problems commonly encountered are those associated with androgen excess or lower levels. Although the level of total testosterone is useful when grossly abnormal, it may be unhelpful in patients with mild or moderate abnormality. In these patients SHBG, LH and FSH measurement are useful. Other estimations, which are also useftd in differential diagnosis, include plasma levels of prolactin, oestrogens, human-chorionic gonadotrophin, (hCG) and adrenal androgens. Thyroid function test may be indicated in problems such as gynaecomastia. 31 University of Ghana http://ugspace.ug.edu.gh 2.11. Testosterone levels associated with problems in infancy. Impaired testosterone formation is one o f the causes of ambiguous genitalia in males and can be due to a defect in biosynthesis. This can be a deficiency of any of the enzymes involved in the synthesis of testosterone, Leydig cell hyperplasia and others. A lower basal testosterone with elevated gonadotrophin and a poor response to hCG, suggests either Leydig cell hypoplasia or biosynthetic defect (Savage, 1982 and Schwatz et al., 1981). Measurement of androstenedione, DHA, 17-OHP and progesterone help to define the enzyme block (Forest, 1981) A normal basal testosterone with a clear and brisk rise following hCG, stimulation suggested either 5 a reductase deficiency or partial end organ insensitivity. These types of patients appear like females until later in life (Migeon et al., 1981). Plasma testosterone and 5 a - dihydrotestosterone ratio, and the ratio of urine 5(3-to 5a metabolites may aid in this differential diagnosis, (Peterson et al., 1977 and Saenger et al., 1978). It should be noted that these ratios are lower in infancy, (Pang et al., 1979 and Saenger et al., 1978). Testosterone and 5a-DHT ratio are indicative of complete insensitivity, or hypothyroidism, ( Imperato Mc-Ginley et al., 1982 and Mauvais-Jarvis, 1981). The measurement of 5a-reductase activity, (Seanger 1979,Kuttenn et al., 1979 and Imperata-Mc Ginley 1980), and androgen receptors in genital skin samples helped to make definitive diagnosis, (Migeon et al., 1981, Griffin and Wilson., 1977). In conditions like Klinifelter's, Down’s, Nuonan’s and male Turner’s syndromes, prepubertal gonadotrophins were low due to partial androgen insensitivity (Lee et al., 1980). These later on in life manifested as delayed puberty after presenting as micropenis in the neonatal stage. 32 University of Ghana http://ugspace.ug.edu.gh Plasma testosterone increased after hCG stimulation in patients with palpable gonads which indicates the presence of functioning testicular tissue. Lack of response indicated the possibility of anorchia ie the vanishing testis syndrome (Savage, 1982 and Spitz, 1983) 2. 12 Testosterone levels in problems associated with prepubertal and pubertal stages. In precocious puberty testosterone, LH, FSH, 17-OHP and adrenal androgens, plus prolactin, p-hCG and alpha-fetoprotein (AFP) are measured. If the hormones are normal then it suggests idiopathic precocious puberty. Hyperprolactinaemia or diabetes insipidus suggest a hypothalamic lesion (Ismail et al., 1986). Increased androgens with low LH levels may be due to excess androgen production due to CAH, adrenal or Leydig cell tumour. In premature adrenarche , development of pubic hair with or without axillary hair as early as 5 years of age in girls and boys monitoring of androgen levels periodically will ensure that adrenarche is not the expression of an androgen producing tumour, (Korth-Schultz et al._±_\ 976). By estimating plasma levels of adrenocortical androgens, DHA, DHA-S and androstenedione can show whether hair development is appropriate, as these are readily suppressed by dexamethasone. In delayed puberty ie when signs of testicular enlargement do not appear by the age of 15yrs of age or so (Ducharme and Collu, 1982), when due to primaiy hypogonadism, plasma testosterone is low with an increase in circulating gonadotrophins. Stimulation with hCG causes little or no rise in circulating testosterone levels. 33 University of Ghana http://ugspace.ug.edu.gh In adults impotence in about 90% of patients has an exclusively psychogenic cause, but erectile potency has been found to be of a complex integrated process, which requires healthy physyche, a functionally intact vascular system, neurological and endocrine systems. Evaluation of all these factors should be done before psychogenic impotence is confirmed (Cooper et al., 1970, Spark et al., 1980 and De Mowbray, 1984). In its investigation drug history is necessary as some drugs decrease potency eg central nervous system depressants and monoamine oxidase inhibitors, anti hypertensives e.g. clonidine and some diuretics eg spironolactone; aestrogens and anti androgens. Investigations usually performed are plasma testosterone, LH, FSH and prolactin estimations and Thyroid Function tests. In hypogonadotrophic hypogonadism and hyperthyroidism, plasma testosterone may be very high due to increase in SHBG with free testosterone being low. 2.13. Testosterone level in gynaecomasia. In gynaecomastia there is excess glandular enlargement of the mammary gland in males. If seen in the neonate, it is attributed to the action o f placental oestrogens. At puberty it could also appear, and at this stage was usually due to imbalance of oestrogen/androgen production, (Large et al., 1979 and Moore 1984). This condition was usually associated with low testosterone or low normal testosterone with high LH. This was also seen in gonadal insufficiency or possibly in hCG secreting tumours (Carlson, 1980). Increased LH, testosterone and oestradiol suggested partial androgen resistance with genital ambiguity, or isolated gynaecomastia, or an hCG secreting tumour (Larrea et al., 1978). 34 University of Ghana http://ugspace.ug.edu.gh 2.14 Testosterone levels in infertility In infertile couple De Mowbray (1984) and Ross (1983), found the occurrence to be 10%. In these the abnormality in both the males and females were important. Male fertility depends on normal spermatogenesis and the status of the seminal tract and fluids, as well as FSH and androgens. In the laboratory investigations, drag history was important as some drugs may inhibit spermatogenesis. These include cytotoxic, anti malarial drugs, monoamine oxidase inhibitors, nitrofurantoin and sulphasalazine (Bateman 1980, and Buchanan and Davis., 1984). After semen and chromosomal analysis, and testicular biopsy have been investigated endocrine tests were also done. These were mainly FSH, LH testosterone prolactin and at times 17-OHP. The single most important basal test was FSH (Wu et al., 1981 and De Kretser 1979). Basal FSH rises when the sperm density is below 1-2 million / ml. A normal FSH and testosterone in an azoospermic patient is diagnostic of abstraction in the duct system that is treatable. Patients with germ cell hyperplasia in the seminiferous tubules have elevated FSH but normal testosterone. In primaiy or secondary hypogonadism, testosterone and LH may be normal. A markedly elevated serum testosterone (>35nmols/L) was indicative of occult hyperthyroidism and requires thyroid function tests, (O’Brien 1982). Raised testosterone levels with equally elevated LH suggests end organ insensitivity and androgen may be limited to a few target cell / organs (Aiman et al., 1979 and Schulster et al., 1983). In these cases FSH was at times elevated. 35 University of Ghana http://ugspace.ug.edu.gh Hyperprolactinaemia in men caused functional suppressing of testosterone, LH and FSH concentrations. In hirsuit patients a high testosterone concentration more than twice the upper limit in women, was an indication of an androgen-producing tumour. Testosterone was also very high in testosterone secreting ovarian tumours while polycystic ovary syndrome exhibit signs of mild to moderate androgen excess (Yen 1980 and Vermeulen, 1984). In men with testicular tumours, testosterone levels were subnormal or normal with raised gonadotrophin levels, (Fossa et al., 1980, Aiginger et al., 1981, Berthelsen et al., 1983 and Cutfield, 1983). 2.15. Testosterone levels in stress. Serum testosterone decreases temporarily in response to severe stress situations eg surgery, (Oyama and Kudo, 1972, Nakashima et al., 1975 and Aono et al., 1976), respiratory failure (Sample et al., 1980), and after unaccustomed exercise (Dessypris, 1976). In anorexia nervosa, there is low plasma testosterone and gonadotrophins during the acute phase, (Palmer et al., 1975). During weight gain the gonadotrophins returned to normal but testosterone remained low to low normal for some time, (McNab and Hawton., 1981). 2.16. Plasma testosterone in Renal and liver diseases In chronic renal failure there was decreased libido and potency with infertility at times. Plasma testosterone was low normal or decreased but LH was increased, (De Kretser et al., 1979, Holdsworth, et al., 1977, Van Kammen, et al., 1978). These changes correlate with the degree of renal failure. Plasma FSH may be normal or raised, with impaired spermatogenesis, (De Kretser 1979). These conditions may be reversed after transplantation but unaffected by dialysis, (Cowden, et al., 1978, Lim et al., 1979 and Holdsworth et al., 1978) 36 University of Ghana http://ugspace.ug.edu.gh In chronic alcoholic males with or without liver disease there was often hypogonadism with oligospermia, or azoospermia, and sometimes oestrogenisation with gynaecomastia. (De Kretser et al., 1979, Boyden and Parmenter, 1983 and, Van Thiel and Lester, 1979). Plasma testosterone levels decreased but SHBG and LH increased. Recovery of sexual function may occur in some but not all after a long period of abstinence, (Van Thiel and Lester, 1979). In coeliac disease there was increase in testosterone, increase in SHBG and free testosterone. There was also an increase in plasma LH concentrations suggesting resistance to testosterone action. In vasectomy there was no evidence of significant alteration in testicular endocrine function (Whitby, et al., 1979). In testosterone replacement therapy the measurement of testosterone was important in order to optimise therapy. 37 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 3.0 Materials and Methods. 3 .1 Specimen Collection. Healthy volunteers ages between llyears to 70years of males were selected by questionnaire. People selected should have body mass index (BMI) of less than 30, cholesterol within the reference range, not on drugs, no surgery within the past 6 months. Other details can be seen on the specimen questionnaire in the appendix. Glass tubes were used throughout in preference to plastic tubes because Ismail et al., (1986) found out that steroids readily adsorbs on to plastic surfaces. The glass tubes were washed thoroughly in distilled water and drained dry at temperatures below 50°C to prevent activation of sites on the glass tubes. One hundred and twenty volunteers between ages 11-70 years, (twenty from each age group) comprising of bank workers, hospital workers, office workers, school children and friends. (Cross sectional) Five mililiters of blood was drawn from each of the 120 volunteers from the anti-cubital vein, with the use of a tourniquet between the hours of 7am and 9 am whilst sitting down. The blood specimens were put into plain glass specimen tubes without any anticoagulant. These were separated within 2 hours of collection after clot retraction, by centrifiigation in a horizontal rotor head centrifuge at l,200g relative centrifugal force for 5minues. The separated sera were pipetted into small tubes (75x10mm), stoppered and stored at 20°C in a freezer. This is because there is increase in testosterone concentration if blood was left for 24 hours at room temperature if blood is left at 40°C for the same time before separation the increase is minimal but not abolished. (Ismail et al 1986). 38 University of Ghana http://ugspace.ug.edu.gh 3.2.0. Testosterone Assay. 3.2.1 Testosterone Reagent Preparation EIA testosterone kit was bought from Biosource Europe. S. A. Microtitre plate with 96 wells coated with goat anti rabbit IgG was ready for use. The O.Ong/ml standard in human serum and preservatives was reconstituted with 1ml. of distilled water.The standards with concentrations 0.0, 0.15, 0.30, 1.30, 4.7 and 15ng /ml were all reconstituted with 0.5ml distilled water and mixed well. The two controls with concentrations 0.57+ 0.14 and 7± 0.6ng/ml were reconstituted with 0.5ml distilled water and mixed well. 0.2mls the concentrated Testosterone - HRP conjugate in phosphate buffer with preservative was pipetted into each of the three (3) conjugate buffer vials. Five microliters o f the 20% of Tween 20 was diluted with I L of distilled water. One hundred of tetramethyl benzedine (the chromegen) was pipetted into each of the two vials of substrate buffer containing H2O2 in acetate/citrate buffer. One vial of 6ml.of stop solution containing 1.8N H2SO4 was ready for use. 3.2.2 Testosterone methodology. All pipetting were done using Variable Volac High Precision Pipette. All sample controls standards and reagents were allowed to attain room temperature. The required number of microwell strips needed were selected and fitted into the holding frame. Fifty microliters of each standard, control and sample were pipetted into the appropriate wells. Two hundred microliters of anti testosterone antibody conjugated with horse radish peroxidase in buffer was pipetted into each well. All wells were incubated for 2 hours at room temperature on a Denley Wellscan automatic horizontal shaker set at 700± 100 revolutions per minute (RPM). The wells were then washed by aspirating the liquid from each well, and then washing them by dispensing 0.4ml of 20% Tween 20 washing solution. The washing step was repeated two times. Two hundred microliters of the 39 University of Ghana http://ugspace.ug.edu.gh freshly prepared revelation solution containing tetra methyl benzedine was pipetted into each well immediately following the washing step. The wells were incubated for 30min. at room temperature, avoiding direct sunlight on the Denley Wellscan automatic horizontal shaker. Fifty microliters of hydrochloric acid stopping reagent was pipetted into each well and mixed gently. The absorbance was read at 450nm within I hour on the Denley Wellscan reader. Calculation For each standard, sample or control, the B/BO, 100 OD (standard, sample or control-) x 100 OD (zero standard) Using either linear-linear or semi log graph paper, the (B/BO x 100) values for each standard point were plotted as a function of the testosterone concentration o f each standard point. By interpolation of the samples (B/BO x 100) values, the testosterone concentrations of the samples and the controls were obtained from the reference curve. AD values were converted to nmol/L by using a convertion factor of 3.47. All results were multiplied by 3.47. 33.0. LH Assay. 3.3.1. Reagent Preparation For LH. Six LH standards with concentrations 0,4,12,36,90,180 IU/mL were reconstituted with 0.8ml of distilled water per vial They were allowed to stand for 15 mins. The vials were inverted several times to ensure that the contents were thoroughly dissolved.One bottle of sample diluent was ready for use. One bottle of enzyme conjugate was ready for use.One bottle of substrate solution A was ready for use. One bottle of substrate solution B was ready for use. One bottle of stop solution was ready for use. 40 University of Ghana http://ugspace.ug.edu.gh 3.3.2 Methodology For LH. LH Kit was purchased from World Wide Diagnostics. Ridgefield. USA. All reagents were brought to room temperature (20-30° C) Fifty microliters of standards samples, and controls were pipetted using volac variable pipette and added into each well respectively containing LH antibody raised from rabbit. Seventy-five microliters of sample diluent was added to all wells and mixed gently. The microwells were placed in a humidified box and incubated at 37°C for 30 minutes by shaking on a Danley Wellscan automatic shaker. The contents of the wells were decanted and washed 6 times with running tap by filing each well and inverting the plate vigorously to get rid of residual water droplets. Blot the rim of each well on an absorbant paper. One hundred microliters of Enzyme Horse radish peroxidase conjugated with anti LH antibody was pipetted into each well and mixed gently. The wells were then incubated for 30mins. Step 5 was repeated. Fifty microliters of substrate solution A was dispensed into each well followed by substrate B. The wells were mixed gently and incubated for 15mins. Fifty microliters of stop solution was added to each well to stop the reaction. The Denly Wellscan EIA reader was zeroed with blank control well and all wells were read at 450nm within 30min. The EIA reader calculated the concentrations automatically. 3.4.0. FSH Assay. 3.4.1. Reagent preparation for FSH. FSH kit was purchased from World Wide Diagnostics Ridgefield, USA. FSH Microwell plate coated with monoclonal antibody specific for FSH was ready for use. Six FSH standards with concentrations 0.0, 0.6,4,12,36,90 and 180miu/lml were reconstituted by adding 0.8ml of distilled water per vial. The vials were allowed to stand for 15min.The vials were then inverted several times to ensure that the contents were thoroughly dissolved. The zero standard was ready for use. 41 University of Ghana http://ugspace.ug.edu.gh One bottle enzyme conjugate was ready for use One bottle each of enzyme conjugated polyclonal antibody against alpha subunit of hCG, substrate solution A, substrare solution B, stop solution IN. HCL diluted with 200 mis distilled water were ready for use. 3. 4.2. FSH Methodology. All pipetting were done using Variable Volac High Precision Pipettes. All reagents were allowed to reach room temperature and the microwells containing FSH antibody raised from rabbit were placed on the holder. Fifty microliters of serum, standards or controls were pipetted into each respective well. One hundred microliters of Enzyme conjugate was also pipetted into each well and mixed thoroughly by swirling the plates on the flat bench for 10 seconds. The plates were incubated at 37°C for 60 mins with shaking. The contents of the microwells were poured into the sink, followed by rinsing the wells 5 times with running tap water. The plates were taped on absorbent papers to get rid of residual droplets. Fifty microliters of substrate solution A was added into each well followed by the addition o f 50p.l of substrate solution B in each well and mixed The wells were incubate at room temperature for 5mins. The colour reaction was stopped by adding 50jxl o f the stop solution into each well and gently mixed for 10 seconds. The EIA. Reader was blanked with blank the control well, and all wells were read at 450nm. 3.5.0. SHBG Assay. 3.5.1. Reagent preparation for SHBG. SHBG Kit was bought from Bio Source Europe S.A. Microtitre plate with 96 wells on a plate coated with mouse monoclonal SHBG antibody packed in a laminate bag was ready for use.Assay buffer of 80mls was ready for use. 42 University of Ghana http://ugspace.ug.edu.gh SHBG standards (0.0,4.0,16, 65 and 260 nmol/L) were ready for use. Control standard was ready for use. Horseradish peroxidase conjugated mouse monoclonal SHBG antibody was diluted 1:100 with assay buffer before use. Five mis o f Tween 20 was diluted to 1 L with distilled water. TMB substrate solution and stopping reagent I N HCL was ready for use. Intra-assay coefficient o f variation was between 3.0 to 8.6% wit inter assay CV of between 7.2 + 0 11.6% 3.5.2. SHBG Methodology. All pipetting was done using Variable Volac High Precision Pipette. All reagents were left to reach room temperature before use. Controls and standards containing 0.0, 4.0,16,65,260nmol/L were ready for use. Wells to be used were arranged on the plate. Standards, control sera and samples were diluted with 1:20 with assay buffer. One hundred microliters of assay buffer was pipetted into appropriate wells. Twety-five microliters of diluted standards, control sera and samples were pipetted into appropriate wells at 10 seconds interval. The plates were covered and incubated for 30mins at room temperature with shaking. The wells were aspirated and washed 3 times with 300(a.l of washing solution. (20 % of Tween 20). At 10 seconds intervals lOOjil containing mouse monoclonal SHBG antibody conjugated with horseradish peroxidase was pipetted into all wells. The plates were incubated for 15 minutes at room temperature. The reaction was stopped by adding 100jnl of TMB substrate solution was added to each well. The plates were covered and incubated for 12 minutes at room temperature. The reaction was stopped by adding 100(il o f stopping solution, 1:100 dilution of IN HC1 to each well at 10 seconds intervals, and the plates were shaken gently to mix. The absorbance was measured at 450nm on the Denley 050 Wellscan plate reader. The EIA reader automatically calculated the values. 43 University of Ghana http://ugspace.ug.edu.gh CALCULATION: The absorbance value of the zero standard was subtracted from the absorbance value of standards, control and samples. A standard curve on log-log graph paper by plotting absorbance values of standards against appropriate SHBG concentrations. The SHBG concentrations for controls and samples can be read off the graph, but the Denley 050 Wells Scan calculated the concentrations automatically. Statistical Evaluation. Normal distribution o f all hormone variables was tested by the Kolmogorov-Smimov test and natural logarithmic transformation was performed on all hormonal variables. Bivariate correlation analysis was performed using age and the various hormonal parameters, respectively. AD subjects were sub grouped by age decades and using Anova and Bonferroni post hoc test tested means of all hormones. Multiple regression analysis was applied for evaluation of joint effects o f age on serum levels of testosterone, FSH, LH and SHBG. The lowest level of the male sex hormones was determined in young men (21-30 years) and used as the cut-off for normal values for adults. In order to find the age dependent variations, the mean o f the different age groups were compared to that o f the younger men. Two-sided p< 0.05 was considered significant. Computations were performed using Statistical Package o f Social Studies, (SPSS), Chicago, IL USA.) The ranges are given as mean ± SEM. 44 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4.0. RESULTS: The result are shown in the different graphs 40 0 i 11-20 21-30 31-40 41-50 51-60 61-70 Age Groups (years) Fig 4.1 Mean levels of serum testosterone with age 45 Mean Testosterone levels(nmol/L) University of Ghana http://ugspace.ug.edu.gh Std. Dev = 5.91 Mean = 33.8 N = 64.00 22.5 27.5 32.5 37.5 42.5 47.5 52.5 57’ .5 25.0 30.0 35.0 40.0 45.0 50.0 55.0 Testosterone Levels. Fig 4.2 Frequency distribution of testosterone Levels. 3 1 9 3 31 3 . 4 4 3 . 5 6 3 6 9 3 81 3 . 9 4 4 . 0 6 Log transformed testosterone levels. Fig 43. Frequency distribution of log transformed testosterone levels. 46 Frequency University of Ghana http://ugspace.ug.edu.gh Table 4.1. Means ranges and percentage change of serum Testosterone. Age groups Means Ranges 95% Confidence % Range Change. 11 -20yrs. 18.66nmol/L 18.66+3.50nmol/L 11.6-25.66nmol/L “ 21-30yrs. 34.02nmol/L 34.02±.99nmoI/L 32.02-36.0nmol/L 45.1 31-40yrs. 32..26nmol/L 32.26+1.20nmol/L 29.50-34.66nmol/L 5.2 41 -50yrs. 33.47nmol/L 33.47+1.07nmoI/L 31.33-35.50nmol/L 3.6 51 -60yrs. 32.29-13nxnol/L 32.29+1.03 nmol/L 30.24-34.36nmol/L 3.5 61-70yrs. 30.13nmoI/L 30.13±2.07nmol/L 25.99- 34.27nmol/L 6.6 47 University of Ghana http://ugspace.ug.edu.gh The analysis for testosterone age groups by decades showed the different ranges as follows: The 11 to 20 years age group had a mean o f 18.7±3.5 SEM giving a range o f 11.7 to 25.7nmol/L The 21 to 30 years age group had a mean o f 34.0nmol/L with a range o f 34.0+0.99 SEM giving an actual range o f 32.02- 36.0nmol/L.There was an increase o f 45.1%from the previous group. The 31 to 40 year age group had a mean o f 32.3±1.2 SEM that is the 29.50-34.7nmoI/L. There was a decline o f 5.2% from the previous group. The 41 to 50 age group had a mean o f 33.5nmol/L with a range o f 33.5±1.0nmol/L SEM that is 31.5 to 35.5 nmol/L. There was an increase o f 3.6% from the previous group. The 51 to 60 age group had a mean o f 32.3 nmol/L a range o f 32.3±1.03nmol/L SEM that is 30.24 to 34.36snmol/L. There was a decline o f 3.5% from the previous group. The 61 to 70 age group had a mean o f 30.13nmol/L with a range o f 30.13±2.07nmoI/L SEM that is 25.99 to 34.27snmol/L. There was a decline o f 6.6% from the previous group. Statistical evaluation gave a probability value o f p< 0.000 for the 11-20 years age group showing that those values were significantly different from the others. This was because it included the pubertal ages that is 11 year to 20 years, whose testosterone levels were much lower. The decline in total testosterone started after age 50 years. The percentage decline from the age of21-70 years was 11%. 48 University of Ghana http://ugspace.ug.edu.gh This shows that Ghanaian males have a lower decline per decade in total testosterone than their Caucasians counterparts. The total testosterone range quoted, by the kits was 10.4 to 34.7nmol/L was lower than the testosterone range (Ghanaian males) in this study. The percentage decline per decade starting from age 31-70 years was 4.6%. The decline in total testosterone started after age 31 years, but a more pronounced decline started at the age o f 60years. From the 20years age group to the 70years age group, the total decline was 11%. The distribution curve obtained for ages 20-60 years o f age gave a mean o f 33.8nmol/L with a range o f 33.8±1.96 nmol/L SD, 95% confidence limit o f 33.8+0.84nmol/L SEM, (32.3-35.3 nmol/L). The geometric mean gave a value o f 33.39±1.17nmol/L giving a range o f 31.05- 35.73nmol/L, with a 95% confidence range o f 31.35-35.13nmol/L. The distribution curves in both the untransformed and the log-transformed curves were positively skewed. Intra batch assay CV was 1.76% with an inter batch assay CV o f 1.70. The effect o f storage was investigated and the findings were as followed. The baseline Value was 24.68nmol/L and after two months when the estimations were done the results obtained was 24.32nmol/L, which was not statistically significant. The present study used Randox quality control sample o f known values to check the precision o f the results. 49 University of Ghana http://ugspace.ug.edu.gh Randox values Values obtained in this study Testosterone Level. 1 .4.8nmol/L 3.9nmol/L Testosterone Level.2. 16.8nmol/L 16.2nmol/L A previous testosterone values done obtained in both Ghanaian males and females using another EIA (Fenzia ELA obtained from Orion Diagnostics from Finland) gave a mean value o f 36.7nmol/L with a range 36.7+1.19nmoI.L SEM) the 95% confidence limit was 34.4-39.1 nmol/L, (Unpublished observation.). The differences between the two EIA values may be associated with the age groups worked on. The present study’s age group was 20 to 70while the previous study’s age group was 18-46 year, which was in the very active age group as indicated in the present study. 50 University of Ghana http://ugspace.ug.edu.gh 51 Mean SHBG levels (nmol/L) University of Ghana http://ugspace.ug.edu.gh 1 S I d . D e v * 1 7 - 6 3 M e a n = 3 8 . 9 N = 6 2 0 0 4 5 . 0 5 5 . 0 6 5 . 0 7 5 . 0 8 5 . 0 SHBG Levels Fig 4.5. Frequency distribution of SHBG Levels. S t d . D e v = 4 8 M e a n - 3 . 5 6 N = 6 2 . 0 0 2 . 3 8 2 . 6 3 2 . 8 8 3 13 3 . 3 8 3 . 6 3 3 . 8 8 4 . 1 3 4 . 3 8 Log Transformed SHBG levels Fig 4.6. Frequency distribution of Log Transformed SHBG Levels. 52 Frequency Freauencv University of Ghana http://ugspace.ug.edu.gh Sex hormone binding globulin. Table 4.2. Means, ranges and percentage change of SHBG. Age Groups Mean Values Ranges 95% Confidence % Change 1 l-20years 36.98nmol/L 36.98±5.34nmol/L 26.30-47.88nmol/L 21-30 38.65nmol/L 38.65±9.9nmol/L 36.67-40.53nmol/L 4.3 31-40 38.29nmol/I 38.03+5,46nmol/L 27-37-49-2nmol/L 3.6 41-50 37.03nmol/L 36.03+4.32nmol/L 28.27-45.2nmol/L 3.2 51-60 36.29nmol/L 36.29+4.32nmol/L 27.65-44.93nmol/L 2.0 61-70 36.74nmol/L 36.74+7.96nmol/L 20.82-52.66nmol/L 1.2 53 University of Ghana http://ugspace.ug.edu.gh The results obtained for sex hormone binding globulin with age groups show the following: Ages 11 to 20 years had a mean o f 36.98nmol/L with a range o f 36.98±5.34nmol/L (26.30 to 47.66nmol/L) Ages 21-30 years had a mean o f 38.65nmol/L with a range o f 38.65±3.34nmol/L (SEM). This will give a range value o f 31.97 - 45.33nmol/L. There was an increase o f 4.3% from the previous group. Ages 31-40 years had a mean o f 38.29nmol/L with a range o f 38.29±5.46nmol/L (SEM). This will give the range values o f 27.37 to 49.21nmol/L. There was a decrease o f 3.6%. from the previous group. Ages 41-50 had a mean o f 36.29nmol/L with a range of36.29±4.32nmol/L (SEM). This gave actual values o f 27.65 to 44.93nmol/L. There was a decrease o f 3.2% compared to the previous age group Ages 51-60 years group had a mean value o f 36.29±4.32nmoI/L.This gave a range o f 27.65-44.93nmol/L. There was a decrease o f 2.0% from the previous group. Ages 61-70 had a mean o f 36.74nmol/L with a range o f 36.74±7.96nmol/L (SEM) with an actual range o f 20.82 to 52.66nmol/L.There was a marginal increase o f 1.2% from the previous group. From these it can be seen that age group 11-20 had the highest SHBG level with the age group 61-70 having the overall lower values 36,74±5.34nmol/L (SEM). Active age groups used by the kits for the establishment o f the ranges were 20-60years.The over all declines in concentration was 6.1%. 54 University of Ghana http://ugspace.ug.edu.gh The distribution curve was also taken from ages 20-60 as this is the age group considered to be the active age group, gave a mean o f 38.9nmol/L with a range o f 38.9 ± 2.23 nmol/L SEM, with a range o f 34.4 - 43.36nmol/L The reference range quoted by Tietz was 20-45nmol/L. The lower range obtained in this study was higher than the 20nmol/L quoted by Tietz. The lower ranges obtained were between 20.82 to 36.67nmol/L with the higher ranges between 40.53nmo/L - 52.66nmol/L. Coefficient o f variations were 1.01% for intra batch CV, and 1.22% for inter batch CV. The probability values between the groups was p< 0 .99 which showed that the differences between the different age groups were not significant. There was a slight increase in SHBG after the 6th decade. The percentage increase in concentration compared to the 50 to 60 years age group was only 1.2%. The frequency distribution curve was positively skewed. The value obtained were 34.44- 43.37nmol/L The log- transformed values for SHBG were 34.99±1.79nmol/L while the 95%Confidence interval values were 31.41-38.57nmol/L and this compares with the values quoted by the department. 55 University of Ghana http://ugspace.ug.edu.gh 11-20 21-30 31-40 41-50 51-60 61-70 Age Groups (years) Fig 4.7. Mean levels of LH with Age 56 University of Ghana http://ugspace.ug.edu.gh S. G 1 0 . 0 1 5 . 0 2 0 . 0 2 5 . 0 3 0 . 0 3 5 . 0 4 0 . 0 LH Levels Fig 4.8. Frequency distribution of LH levels. S t d . D e v = .58 M e a n = 2 . 0 6 N = 6 0 . 0 0 . 7 5 1 . 2 5 1 . 7 5 2 . 2 5 2 . 7 5 3 . 2 5 3 . 7 5 1 . 0 0 1 . 5 0 2 . 0 0 2 . 5 0 3 . 0 0 3 . 5 0 Log LH Levels Fig 4.9. Frequency Distribution of Log Transformed LH Levels 57 Frequency University of Ghana http://ugspace.ug.edu.gh Table 4.3. Showing LH means, ranges and increases or deceases Ages Mean Range 95% Confidence %Change 11-20 6.72UL 6.72+2.49U/L 1.74-11.70U/L 21-30 10.44UL 10.44+2.37U/L 5.70-15.18U/L 35.6 3140 8.25U/L 8.25+1.13U/L 5.99-10.5U/L 21.1 41-50 9.30U/L 9.30+.99U/L 7.32-11.28U/L 11.3 51-60 9.24U/L 9.24+1.91U/L 5.42-13.06U/L 8.2 61-70 6.96U/L 6.9+2.22U/L 2.52-11.40U/L 24.2 58 University of Ghana http://ugspace.ug.edu.gh The LH Results showed that the 11 to 20 age group had a mean o f 6.72nmol/L with a range o f 6.72±2.49 (SEM) giving a range o f 1.74 to 11.70nmol/L. The 21-30 years age group had a mean o f 10.44±2.37 (SEM) giving a range o f 5.70 to 15.18nmol/L. There was an increase o f 35.6% from the previous age group. The 31-40 years age group had a mean value o f 8.25±1.13 (SEM) giving a range o f 5.99 to 10.51nmoI/L. There was a decrease o f 21.1% from the previous age group. The 41-50 years age group had a mean o f 9.30+0.99 (SEM) giving a range o f 7.32 to 11.28nmol/L. There was a decrease o f 11.3% from the previous age group. The 51- 60 years age group had a mean o f Q.24±1.91 giving a range o f 5.42 to 13.06nmol/L. There was a decrease o f 8.2% from the previous age group. The 61 - 70 years age group had a mean o f 6.96+2.22 giving a range o f 2.52 to 11.40nmoI/L. There was a decrease o f 24.2% from the previous age group. It can be seen that all the different age groups had higher lower reference range (1.74 U/L) and a higher, higher limit respectively (15.18U/L) reference values. The overall decline from the 21to 30years age group to the 60 to 70years age group was 33.3%. The probability value was p= 0.746 showing that the between group variations were statistically not significant. The frequency distribution curve for the adult range (20-60years o f age gave a 9.4±0. 85 U/L(SEM) with a range o f 7.7-11U/L The log transformed values were 7.86+1.08SEM giving a range o f 5.70-10.02U/L Sensitivity o f the assay was 0.05U/L Coefficient o f variation. The values obtained in this study were higher than the quoted values by the kit (1.5-9.0U/L). 59 University of Ghana http://ugspace.ug.edu.gh The intra batch CV was 4.14% with an inter batch CV o f 3.88%. The LH frequency distribution curve was positively skewed. The mean for LH gave values o f 7.86±1.06U/L with the range o f 5.74-9.98U/L. The values were slightly higher than the quoted values (1-9U/L) The ANOVA value was p =0.746 showing that the between group variations were not significant. Precision studies using Randox controls were as follows Quoted value Obtained values Level 1 0.57U/L 0.88U/L Level 2 68U/L 65.4U/L 60 University of Ghana http://ugspace.ug.edu.gh 10.0 ■ 8.0 ■ 11-20 21-30 31-40 41-50 51-60 61-70 Age Groups (yrs) Fig 4.10. Mean Levels of FSH with Age. 61 Mean FSH levels (U/L) University of Ghana http://ugspace.ug.edu.gh FSH Levels Fig 4.11. Frequency distribution of FSH levels Log FSH Levels Fig 4.12. Frequency distribution of Log Transformed FSH levels. 62 University of Ghana http://ugspace.ug.edu.gh Table 4.4. Means, ranges and percentage change of serum FSH levels. Age Groups Means Ranges 95% confidence %Change 1 l-20yrs 7.63U/L 7.63+2.39U/L 2.85-12.4U/L 21-30 7-18U/L 7.18+1.32U/L 4.54-9.82U/L 5.9 31-40 8.57U/L 8.57+1.89U/L 4.79-12.36U/L 16.2 41-50 6.79U/L 6.79+1.94U/L 2.91-10.67U/L 20.7 51-60 6.39U/L 6.39+0.66U/L 5.07-7.7U/L 5.9 61-70 5.94U/L 5.94+1.32U/L 3.30-8.58U/L 7.7 63 University of Ghana http://ugspace.ug.edu.gh FSH results show that in the 11-20 years age group, the mean was 7.63U/L with a range of 7.63±2.39U/L SEM with the 95% confidence o f 2.85-12.4U/L In the 21-30 years age group, the mean was 7.18U/L with a range o f 7.18+1.32U/L (SEM) giving a 95% confidence range o f4.54 - 9.82U/L. In the 31-40 years age group, the mean was 8.57U/L with a range o f 8.57+1.89U/L (SEM) giving a 95% confidence range o f4.79 - 12.35U/L (SEM). There was an increase of 1 6.2% from the previous age group. In the 41-50 years age group, the mean was 6.79U/L with a range o f6 .79+1.94 U/L (SEM) giving a 95% confidence range of 2.91-10.67U/L There was a decrease o f 20.7% between this and the previous age group. In the 51-60 years age group, the mean was 6.39U/L with a range o f 6.39+0.66 U/L (SEM) giving a 95% confidence range o f 5.07 - 7.7U/L. In the 61-70 years o f age group, the mean was 5.9U/L with a range o f 5.94+1.32U/L with a 95% confidence range o f 3.30U/L - 8.58U/L. The frequency distribution curve gave a value o f 7.7+0.82 U/L (SEM) with a 95% confidence interval o f 6.1±8.6U/L. This value was consistent with the already established value o f 1-14U/L quoted by the kits. The percentage decline between the 20-70 years age group was 17.3% 64 University of Ghana http://ugspace.ug.edu.gh The frequency distribution curve was positively skewed with a mean value o f 7.7±0.82U/L and a range o f 6.1-9.34U/L. The logarithmic transformed values were 6.31± 1.08U/L with a range o f 4.15-8.47U/L. These values were similar to the range quoted by the Kit (1- 14U/L). The sensitivity o f the assay was 0.05U/L Precision studies using Randox controls were as follows: Quoted values Obtained values Level 1 0.77 U/L 1.03U/L. Level 2 103 U/L. UOU/L. Coefficient o f variation Lntra batch assay CV = 4.1%. Inter batch assay CV= 2.62%. The frequency distribution curve give a value o f 7.7± 0.82 U/L (SEM) with a 95% confidence range o f 6.1* 8.6 U/L. This is similar to what has been quoted for the kit (1-14U/L). 65 University of Ghana http://ugspace.ug.edu.gh 11-20 21-30 31-40 41-50 51-60 Age Groups □ TESTO BSHBG DLH DFSH I Fig. 4:13 Composite figure of ail the Gonadal Hormones Studies and SHBG. University of Ghana http://ugspace.ug.edu.gh 100.0 90.0 80.0 ♦ ♦ 70.0 60.0 50.0 40.0 30.0 ♦ * * ♦ 20.0 10.0 0.0 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 Testosterone levels Fig 4.14. Correlation between Serum Testosterone and SHBG levels. 67 SHBG levels University of Ghana http://ugspace.ug.edu.gh Testosterone levels Fig. 4:15. Correlation between Serum LH and Testosterone Levels. 68 LH levels University of Ghana http://ugspace.ug.edu.gh 45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 30.0 40.0 70.0 Testosterone levels Fig. 4:16. Correlation between Serum FSH and Testosterone Levels. 69 FSH levels University of Ghana http://ugspace.ug.edu.gh Table 4.5. Correlations between serum Testosterone and serum SHBG TESTO SHBG TESTO Pearson Correlation 1 .405** Sig. (2-tailed) .001 N 64 62 SHBG Pearson Correlation .405** 1 Sig. (2-tailed) .001 N 62 62 ** . Correlation is significant at the 0.01 level 70 University of Ghana http://ugspace.ug.edu.gh Table 4.6. Correlation between serumTestosterone and serum LH TESTO LH TESTO Pearson Correlation 1 .051 Sig. (2-tailed) .703 N 64 59 LH Pearson Correlation .051 1 Sig. (2-tailed) .703 N 59 60 71 University of Ghana http://ugspace.ug.edu.gh Table 4.7. Correlation between serumTestosterone and serum FSH TESTO FSH TESTO Pearson Correlation 1 -.049 Sig. (2-tailed) .703 N 64 62 FSH Pearson Correlation -.049 1 Sig. (2-tailed) .703 N 62 63 72 University of Ghana http://ugspace.ug.edu.gh Table 4.8.CAMPARISON BETWEEN SERUM TESTOSTERONE One-sample Statistics Std. Error N Mean Std. Deviation Mean TESTO 64 33.839 5.9128 .7391 One-sample Statistics Test Value for Caucasians 17.4 95% Confidence Interval of Difference df Mean Lower Upper t Sig.(2-tailed) Difference TESTO 22.242 63 .000 16.439 14.962 17.916 73 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 5.0. DISCUSSION AND CONCLUSION The study had shown that Ghanaian males have higher testosterone levels than the values quoted by the kits, 300-1000ng/dl (10.4-34.7nmol/L). This confirms the findings o f Ellis and Nynborg (1992), that there are racial and ethnic differences in testosterone concentration. The findings by Ross et al (1986) also supported the idea that blacks have higher testosterone levels than their white counterparts. Ross et al., (1986) also found out that the mean testosterone levels in blacks were 19% higher for total testosterone levels and 21% higher in free testosterone levels. In this study free testosterone levels were not measured but a previous study gave a mean of 17.4nmol/L for total testosterone for Caucasians. The present study gave a mean of 33.8nmol/L for Ghanaian males, all using the same EIA method. This gave a higher 95% confidence range for total testosterone. Dominique et al; (1992) showed age related decline in testosterone starting in the early adult years. The decline started from the third decade. The age group they worked on were healthy men aged 20-60years. Leifke et al., (2000) also showed a continuous and parallel decline o f serum sex hormones and insulin growth factor in healthy men between 20and 80 years o f age starting from the third decade. The Ghanaian males showed that the set point for the decline in testosterone was 60years o f age as shown by the present study. Obesity, as defined by body mass index, and upper body or android obesity, as described by subscapular skin fold, both increase significantly with age. Total and free plasma and serum testosterone levels decreased in obese men. The decline is a continuum observable at any level o f obesity, (Zumoff et al., 1990). 74 University of Ghana http://ugspace.ug.edu.gh These facts did not apply to this study as the males chosen for this study had BMI o f <30kg/m2 which was the accepted BMI for the Leifke et al (2000), study. The present data show a late continuous and parallel decline o f serum sex hormones in healthy males between 41-70 years. Most workers (Dominique et al., 1992) worked on healthy men from ages 20-60 years. Leifke et al (2000) worked on 20-80 years age group. Morley et al., (1997) chose the males between 61-87 years while Barrett-Connor, (1999) worked on men from ages 50-89 years. Ferrini et al (1998) when studying the effect of age on sex hormones chose men aged between 24 and 90 years. All these showed various decline values in testosterone levels with age. Morley et al., (1997) also demonstrated a longitudinal decline in testosterone level with average rate o f decrement o f 1 lOng/dl, (3.8nmol/l) per decade, but the present study had an average decrement o f 1.3nmol/l. In non-obese healthy men, age was inversely correlated with serum levels o f all androgens. Total testosterone levels stayed relatively stable until age 55years as found by Vermuelen et al., (1996). The present study gave 50 years as the age when testosterone declines steadily although there were slight declines starting from age 40 years. Testosterone binds to SHBG, and the levels o f SHBG measured in this study did not parallel the decline in testosterone. Free testosterone was not measured but Zumoff et al., (1990) found out that a fall in plasma total testosterone was accompanied by a parallel fell in free testosterone. In this study the SHBG levels did not parallel the total testosterone levels in all the age groups. The highest testosterone level in the 21-30 years age group was also accompanied by the highest level in SHBG in the same age group. 75 University of Ghana http://ugspace.ug.edu.gh Morley et al., (1997) found that SHBG level increased with age. T he age group taken for that study was from 61-87. Although the age groups used in the present study did not go that far, there was a slight increase in SHBG in the 60-70 years age group. The fact that Ghanaian males have much higher ranges than their Caucasians counterparts, have also been seen in a previous study in which another EIA kit was used. The first EIA method used alkaline phosphates as the enzyme o f label while this present study used peroxidase as the enzyme o f label. In that project the mean was 36.7nmol/L with a range o f 18.7-54.7nmol/L. The 95% confidence limit was 34.4 39.1nmol/L. (Unpublished observation). The differences between the two EIA values may be associated with the age group taken. In the previous work the age group used were 18-46years while in the present study the age group chosen for the establishment o f the present range were 20- 60years. This may account for the difference in the values between the previous study and the present one. Ageing alters the set point for androgen negative feedback control o f gonadotropin acceleration in men. Unchanged or slightly increased LH levels and reduced testosterone production is characteristic o f elderly men. Accordingly age-associated Leydig cell insufficiency leads to a decline in testosterone production, but circulating LH levels do not rise appropriately because the set point for negative feedback is decreased (Winter and Atkinson 1997). This was observed in the Caucasians. The results obtained indicated that the highest testosterone level was found in the age group 21-30 yeas. The same increases were seen in SHBG and LH but FSH decreased. LH is needed for spermatogenesis and the set age for the highest production o f sperm is around age 25 years. At this age the average sperm production is 150 million sperm per testis per day. 76 University of Ghana http://ugspace.ug.edu.gh With this high demand, LH is also the highest in this age group. FSH on the other hand was low. This may be the effect o f the negative feed back mechanism working on the anterior pituitary. In the age group 31-40 years a slight decrease is shown in testosterone, SHBG and LH. This again may be as a result of the negative feed back mechanism. The decrease in testosterone level was only 1.76nmol/L, the decrease in LH was 2.19U/L, with that o f SHBG being 2.91 nmol/L. As these decreases register the FSH increased through the negative feedback mechanism to bring the levels o f testosterone, LH and SHBG up again. There were gradual declines from age 40years to 70years in FSH, SHBG, testosterone and LH. The set point for decline in testosterone and LH with age in this present study is at 50 years while that o f FSH and SHBG is at 40years. From this age all the four hormones declined up to age 70years. Testosterone had a decline o f 3.885nmol/L, which is 11% between ages 20-70years. SHBG had a decline o f 1.55nmol/L (4.3%), for the same age group while LH had a decline o f 3.58U/L (34%) and that o f FSH was 0.088U/L (1.5%). It has been documented by Morley et al (1997) that LH, FSH and SHBG all increased with age, but there were rather declines in all the hormones measured, (LH, FSH SHBG and testosterone), in this study. The differences may be brought about because o f the differences in the age group for the two studies 61-87 years for Morley’s study and 21- 70years in this study. In this study there was a slight increase in SHBG but not the rest. This may be as a result o f the very low decreases in testosterone, which did not stimulate the feed back mechanism. As the set point for hypothalamus gonadal feed back 77 University of Ghana http://ugspace.ug.edu.gh mechanism decrease with advancing age, the level o f group testosterone was not low enough to stimulate the feedback mechanism to register increases in LH, FSH and SHBG. Vermeulen et al (1996) documented that albumin concentration decrease with age, and if free testosterone is the feedback regulator of plasma testosterone levels, then albumin concentration might be a co-determinant (although evidently, less important than SHBG) o f testosterone levels and contribute to the age-associated decrease in testosterone levels. 78 University of Ghana http://ugspace.ug.edu.gh CONCLUSION Ghanaian males have higher testosterone levels than their Caucasian counterparts. The lowest values obtained were about twice the Caucasian lowest range, but the highest ranges had differences o f less than twice as compared to the lower ranges. It was also shown that testosterone levels decrease with age. The set point for the decrease in Ghanaian males start from ages sixty, although there were slight decreases between the different age groups. There was a decrease in serum testosterone after age 30 years, but there was an increase again after age 40 years. These differences may be as a result o f the small sample size (15), in every age group. As hormonal therapy is usually needed in the elderly, hormonal levels will be more needed from the age o f fifty and above, when hormonal replacement therapy is mostly needed. In the investigation o f the cancer o f the prostate, testosterone levels are needed to monitor prognosis o f the disease and to maximise treatment, as it has been found in this study that Ghanaian males have higher testosterone levels. The racial variations in testosterone levels may be dependent more on socio cultural factors, e.g. diet or stress. Sex hormone binding globulin stayed almost the same level throughout the adult life but the highest level was in the early adult age, which is also the age with highest testosterone level in Ghanaian males. There was a slight increase after age sixty as documented, but this study did not go beyond age seventy to study the trend in the Ghanaian elderly males. Follicle stimulation hormone and LH also decreased with age after the 5th decade in Ghanaian males. This showed that FSH LH did not increase when testosterone is decreased, as would have been the case. This is as a result o f the lowered set point for the hypothalamic pituitary gonadal acceleration in old age. 79 University of Ghana http://ugspace.ug.edu.gh Recommendation More work should be done on a larger population to enhance the trueness o f the different levels in the age groups. This will give a more meaningful range in Ghanaian males. Further work should be done in the ages, fifty years and above as this is the age group in which hormonal replacement therapy is more needed, and at which most men report with problems associated with decreased libido etc. Testosterone and 5a-reductase should be estimated to find their relationship in males without cancer o f the prostate and males with cancer o f the prostate, as 5a -reductase activities have also been found to be higher in black men. Males should also be advised to do some form o f exercises as this will help to increase their testosterone levels and minimize the rampant decreased libido in most elderly men. Advice should also be given by the clinicians to elderly men to try and eat a slightly heavy, well balanced diet not the usual European breakfast, as this helps the men to gain their strength back in all activities (Personal Communication) 80 University of Ghana http://ugspace.ug.edu.gh REFERENCES Aiginger P., Kolbe H., Kuhbock K., (1981). The endocrinology o f testicular germinal cell tumours. Acta Endocrinol; 97: 419. Aiman J, Griffin JE, Gazak JM, (1979). Androgen insensitivity as a cause o f infertility in otherwise normal men. N Engl J Med; 300: 223-7. Ambrosius WT, Compto JA, Bowsler RR,Pratt JH (1998). Relation o f race, age and sex hormone differences to serum leptin concentrations in children and adolescents: Hormone Res; 49 (5b): 240-6. Anderson DC (1998). Sex hormone binding globulin. Clin Endocrinol; 3: 69 Aono T, Kurachi K, Miyata M (1976). Influence o f surgical stress under general anaesthesia on serum gonadotorphin levels in male and female patients. J Clin Endocrinol Metab; 42:144-8. Barragg JM, Makin HLJ, Tranford DJH, Scott DF (1978). Effect o f anticonvulsants andtestosterone and SHBG levels. J Neurol Neurosurg Psychiatr; 41: 913-4. Barrett-Connor E, Von Muhlen, DG, Silverstein D (1999). Bioavailable testosterone and depressed mood in older men: the Rancho Bernardo Study. Journal o f Clinical Endocrinology and Metabolism; 84: 573-577. 81 University of Ghana http://ugspace.ug.edu.gh Bateman DN (1980). Drugs and sexual function. Adv. Drag React Bull; 85: 308-11. Behere HM, Kliesch S, Puhse G, Reissmann T, Nieschlag E (1997). High loading and low maintenance doses of a gonadotropin-releasing hormone. Antagonist effectively suppresses serum luteinizing hormone follicle-stimulating hormone, and testosterone in normal men. J Clin Endocrinol Metab; 82 (5): 1403-8 Berthelsen JG, Engelholm S, Maase H (1983) Serum Testosterone, LH and FSH in patients with testicular cancer before and after radio and chemotherapy. Scand JUrol Nerphrol; 17:287-90. Boyden TW Parmenter RW (1983). Effects o f ethanol on the male hypothalamic pituitary- gonadal axis Endocrine Rev; 4: 389-5. Brown WA, Laughren TP. William B. (1981) Differential effects of neutroleptic agents on the pituitary-gonadal axis in men. Arch Gen Psychiatr; 38:1270-2 Buchanan JF, Davis LJ (1984). Drug induced infertility. Drug In Clin Parm; 18: 1 22-32. Byrd W, Bennett MJ, Carr BR, Dong Y, Wians F, and Rainey W (1998). Regulation of biologically active dimeric inhibin A and B from infancy to adulthood in males. J Clin Endocrinol Metab; 83(8):2849-54. Carlson HE. (1980). Gynaecomasstia. N Engl J Med; 303: 795-9. 82 University of Ghana http://ugspace.ug.edu.gh Carter JN, Tyson J.E, Tolis G, Van Vliet S, Faiman MD, Friesen HG (1978). Prolactin secreting tumours and hypogonadism in 22 men. N. Eng. J. Med; 299: 847-52. Colombani M., Aranda C. Scaglia H. (1998). Recombinant Human Follicle-stimulating Hormone administration increases testosterone production in men possibly by Sertoli cell- secreted non-steroidal factor. J Clin Endocrinol Metab; 83(ll):373-6. Cooper AJ, Ismail AAA, Smith CG (1970). Androgen function in psychogenic and constitutional types o f impotence. Br Med J; 3. 17-20 Corpas E, Harman S M, Blackman MRC (1993). Human growth hormone and Human ageing. Endocrinol; 9 241-8. Cowden AE, Ratchliffe WA, Ratclifle JG, (1978). Hyperprolactinaemia in renal disease. Clin Endocronol; 9: 241-8. Croflon P.M, Illingworth PJ, Groome NP, Stirling HF, Swanston I, Gow S, Wu FC, MeNeilly A, Kelnar C.J (1997).Changes in Dimeric inhibin A and B during normal early puberty in boys and girls. Clin Endocrinol; 46 (1): 109-14. Cummings D, Merriam G (1999). Gro th hormone and growth hormone secretagogues in adult. In: Contemporary Endocrenology: Hormone Replacement Therapy. (Ed. A. W. Meikle) pp. 61-90 Humana Press Inc. 83 University of Ghana http://ugspace.ug.edu.gh Cutfield RG, Bateman JM, Odell WD (1983). Infertility caused by bilateral testicular masses secondary to congenital adrenal hyperplasia (21a-hydroxylase deficiency). Fertil Steril; 40: 809-14. Dani-Haeri J, Oxley J (1982). Reduction o f free testosterone by antiepileptic drugs. Br Med J 284 85-6. De Kretser DM (1979). The effects o f systemic disease on the function o f the testis. Clin Endocrinol Metab; 8 : 487-98. De Kretser D.M (1979). Endocrinology of male infertility. Br Med Bull; 35: 187-92. De Mowbray RR (1984). Male hypogonadism, infertility and impotence In: Milo Keynes W. Fowler PBS Eds. Clinical Endocrinology. London Wm Heinemann. 473-524. Dessypris A, Knoppasalmi K, Adlecreutz H (1976). Plasma cortisol, testosterone, androstenedione and luteinizing hormone (LH) in a non-competitive marathon run. J Steroid Biochem; 7: 33-7. Dominique S, Preziosi P, Barrett Connor E, Marc Roger, Saint-Paul M, Nahoul K, Papoz L, (1992). The Influence o f Aging on Plasma Sex Hormones in Men: The Telecom Study Am J Epidemiol; 135:783-91. 84 University of Ghana http://ugspace.ug.edu.gh Dorgan JF, Judd JT, Longcope C, Brown C, Schatzkin A, Clevidence BA, Campbell WS, Nair PP, Franz C, Kahlel, Taylor P.R (1996). Effect o f dietary fat and fibre on plasma and urine androgens and oestrogens in men, a controlled feeding study: Amer J Clin Nutr; 64 (6b): 850-5. Ducharme JR, Collu R (1982). Pubertal development: Normal precocious and delayed. J Clin. Endocrinol Metab; (b) 11: 57-87. Ellis L, Nyborg H (1992). Racial/Ethnic variations in male testosterone levels: A problem contributor to group differences in health; Steroid. 57: 72-75. Fahmer CL, Hackney AC (1998). Effects of endurance exercise on free testosterone and the binding affinity o f sex hormone binding globulin. Inter J o f Sports Med; 19 (lb ): 12-5. Ferrini R L, & Barrett-Connor E (1998). Sex hormones and age: a cross sectional study o f testosterone and oestradiol and their bioavailable fractions in community dwelling men. America journal o f Epidemiology; 147 750-754. Fitzgerald R.C, Skingle S.J, Crisp AJ (1997). Testosterone concentration in men on chronic glucocorticosteroid therapy. J Roy Coll. Phy London; 31(2): 168-80. Forest MG, De-Peretti E, Bertrand J (1976). Hypothalamic-pituitary-gonadal relationship in man from birth to puberty. Clin Endocrinol; 5: 551-69. Forest MG (1981). Errors o f testosterone biosynthesis. In: The Intersex child. Paediatr Adolese Endocrinol Vol. 8. Basel Karger: 133-55. 85 University of Ghana http://ugspace.ug.edu.gh Fossa SD, Klepp O, Aakyaag A (1980). Serum hormone levels in patients with malignant testicular germ cell tumours without clinical and/or radiological signs o f tumour. Br J Urol. 52: 151. Funder JW, Mercer JE (1979). Cimetidine, histamine H2 receptor antagonist occupies androgen receptors. J Clin Endocrinol Metab; 48: 189-91. Garcia-Pascual IJ, Razan Flores MA (1996). Low dose desmopressin (DDAVP) and blood levels of FSH, LH and testosterone in men. Revista Clinica Espamola; 3: 167-170. Giusti M, Guido R, Valenti S, Giordano G (1999). Serum leptin levels in males with delayed puberty during short-term pulsatile Gn-RH administration. J Endocrinol Invest; 22(lb)6-ll . Goodman HM (1994). Biosynthesis o f androgens. In Basic Medical Endocrinology, 2nd Edition, pp 256. Reven Press. New York. Griffin JE, Wilson JD (1980). The testis. In: Metabolic Control and Disease. 8th ed. P. K. Bondy and L. E. Rosenberg, Eds. Philadelphia, W. B. Saunders Co, pp. 1535-1578. Griffin JE, Wilson JD (1977). Studies on the pathogenesis o f the incomplete forms o f androgen resistance in man. J Clin Endocrinol Metab; 45: 1137-43. Hanning RV (1982). Danazol and is principal metabolites interfere with the binding o f testosterone, cortisol and thyroxine by plasma protein. Clin Chem; 28: 696. 86 University of Ghana http://ugspace.ug.edu.gh Haseltine FP, Ohno S (1981). Mechanism of gonadal differentiation. Science; 211:1272. Henderson B.E, Bernstein L, Ross R.K, Depue RH, Judd H L (1988). The early in-vitro oestrogen and testosterone environment o f black and white; potential effects on male offspring. Br J Cancer; 57: 216-18. Holdsworth S, Aikins RC, De Kretser D.M., (1977). The pituitary-testicular axis in men with chronic renal failure. N Engl J Med; 296:1245-9. Holdsworth SR, De Kretser D.M (1978). A comparison o f haemodialysis and transplantation in reversing the uraemic disturbance o f male reproductive function. Clin Nephrol. 10: 146-50. Howell SJ, Radford JA, Ryder WD, Shalet SM (1999). Testicular function after cytotoxic chemotherapy; evidence o f Leydig cell insufficiency. J Clin Oncol; 17 (5): 1993-8. Imperato-McGinley J., (1983). Sex differentiation, normal and abnormal. Curr Top Exp. Endocrinol; 5: 231-307. Imperato-McGinley J, Peterson RE, Stoller R (1979). Male psuedo-hermaphroditism secondary to 17 p-hydroxysteroid dehydrogenase deficiency. Gender role change with puberty. J Clin Endocronol Metab; 43: 391-5. 87 University of Ghana http://ugspace.ug.edu.gh Imperator-McGinley J, Peterson R.E, Gautier T (1982). Hormonal Evaluation o f a large kindred with complete androgen insensitivity. Evidence for secondary 5-a-reductase deficiency J. Clin Endocrinol Metab; 54: 93141. International Federation o f Clinical Chemistry (IFCC) (1978). Provisional Recommendations on the Theory of Reference Values part 1. Clin Chem. 25: 1506-1508. Ismail A.A.A, Astley P, Burr WA, Wood MCA, Short F, Wakelin K, Wheller M.J, (1986). The role of testosterone measurement in the investigation o f androgen disorders. inBiochem; 23:113-134. Judd HL (1979). Biorhythms of gonadotrophins and testicular hormone secretion In: Krieger D.Ted.: Endocrine rhythms. New York: Raven Press; pp 299-234. Jung R (1984). Endocrinological aspect o f obesity. Clin Endorcrinol Metab; 13: 597-612. Kadioglu TC, Koksal IT, Tune M, Nane I, Tellaloglu, S (1999). Treatment o f idiopathic and post varicoselectomy oligozoospermia with oral tamoxifen citrate. Br J Urol Intemat; 83(6): 646-8. Kaufman JM, Vermeulen A (1997). Declining gonadal function in elderly men. Bailliere’s Clinical Endocrinology and Metabolism; 11.289-309. Kauli R (1976). Cyproterone acetate in treatment o f precocious puberty. Arch Dis Child; 51: 202-8. 88 University of Ghana http://ugspace.ug.edu.gh Kent JR, Hill M, Parlow AF, Bisehoff AJ (1972). Oestrogenic suppression o f the pituitary- gonadal axis. Clin. Pharmacol Ther. 13: 144. Kopelman PG, White N, Pilkington TRE, Jeffcoate SL. (1980). Abnormal sex steroid secretion and binding in massively obese women. Clin Endocrinol. 12:363-69. Korth-Schultz S, Levine LS, New MI (1976). Evidence for the adrenal source of androgens in precocious adrenarche. Acta Endocrinol; 82: 342-52. Koskinen E, Katila T (1997). Effect o f 19-norandrostenololylaurate on serum testosterone concentration, libido and closure o f distal radial growth plate in colts. Actu Vererinaria Scandinavica; 38 (1): 50-67. Kumar R, Biggart JD, McEvoy J, McGowen M (1972). Cyclophosphamide and reproductive function. Lancet; 1: 1212 — 14. Kuttemn F, Mowszowicz I, Wright F (1979). Male pseudohermaphroditism: A comparative study o f one patient with 5a-reductase deficiency and three patients with the complete form o f testicular feminisation. J Clin Endocrinol Metab; 49:861-5. Labrie F, Luu-The V, Lin SX, Larbie C, SimardJ, Breton R, Balanger A (1997). The key role o f 17 beta-hydroxy steroid dehydrogenases in sex steroid biology. Steroid; 62(1): 148-58. Lambert SWJ, Van den Beld, AW, Van der Lely AJ (1997). The endocrinology o f aging Science; 278: 419-424. 89 University of Ghana http://ugspace.ug.edu.gh Large DM, Anderson DC (1979). Twenty-four hour profiles and circulating androgens and oestrogens in male puberty with and without gynaecomastia. Clin Endocrinol; 11: 505-21 Larrea F, Benavides G, Scaglia H, (1978). Gynaecomastia as a familial incomplete male pseudohermaphroditsm Type I: A limited androgen resistance syndrome. J Clin Endocrinol Metab; 46: 961-70 Lawrence IG, Price DE, Howlet TA, Harris KP, Feenally J, Walls J (1997). Erythropoietin and sexual dysfunction, Nephrology, Dialysis and Transplantation. 124:741-7 Lee PA, Mazur T, Danish RK, Mazur T (1980). Micropenis. I. Criteria etiologies and classification. John Hopkins Med J; 146:156-63. Leifke E, Geronoi V, Wichers, Von Zur Muhlem A, Von Buren E, Brabant G (2000). Age- related changes o f sex hormones, Insulin-like growth factor 1 and sex hormone binding globulin levels in men cross sectional data from a healthy male cohort. Clin. Endocrinol (Oxf). 53(6): 689-95 Lim VS, Kathpalia SC, Frohman LA (1979). Hyperprolactinaemia and impaired pituitary response to suppression and stimulation in chronic renal failure: Reversal after transplantation. J. Clin Endocrinol Metab; 48: 10. Longcope C (1996). The effect o f human chorionic gonadotrophin on plasma steroid levels in young and old men. Steroid; 21: 583-589 90 University of Ghana http://ugspace.ug.edu.gh Maes M, Mommen K, Hendrickx D, Peeters D, D ’Hondt P, Ranjam R, De Meryer F, Scharpe S (1997). Components of biological variation, including seasonality, in blood concentrations o f TSH, TT3, FT4, PRL, cortisol and testosterone in healthy volunteers. Clinical Endocrinology; 46 (5): 587-98. Mauvais-Jarvis P, Kutten F, Mowszowiez I (1981). Different aspects o f 5ot -reductase deficiencies in male pseudohermaphroditism and hypothyroidism. Clin Endocrinol. 14:459-69. Meikle AW (1999). Androgen replacement therapy o f male hypogonadism In: Contemporary Endocrinology: Hormone Replacement Therapy (ed. A.W. Meikle), pp. 263-306. Humana Press Inc, Totowa. Messina M, Menieri C, Biflignandi P, Massuchetti C, Novi RE, Molinatti G.M (1983) Antiandrogemic properties of spironolactone clinical trial in the management o f female hirstism. J Endocrinol Invest. 6: 23-7. McNab D, Hawton K (1981) Disturbances o f sex hormones in anorexia nervosa in the male. Postgrad Med J; 67: 254-6. Migeon CJ, Bron TR, Fichman KR (1981). Androgen Insensitivity syndrome. In: The interesex child. Paediatr Adolesc Endocrinol Vol.8 Basel: Karger: 71-202. Mitumura R, Jano K, Suzuki N, Ho Y, Makita Y, Okuno A (1999). Diurnal rhythms o f LH, FSH and testosterone secretion before the onset o f puberty. J Clin Endocrinol and Metab. 84: 29-37. 91 University of Ghana http://ugspace.ug.edu.gh Mokshagundam SL, Minocha A (1997). Does concurrent acute ethanol ingestion during omeprazole therapy aflect pituitary gonadal axis in male subjects? J Toxicology — Clinical Toxicology; 35(1): 55-61 Moore DC, Schlaepfer LV. Paunier L. (1984). Hormonal changes during puberty vs. transient pubertal gynaecomastia. Abnormal androgen oestrogen ratio. J Clin Endocrinol Metab; 58: 492-9. Mowszowicz I, Wright F, Vincens M (1984). Androgen metabolism in hirsute patients treated with cyprotone acetate. J Steroid Biochem; 20: 757-61. Morley J E, Kaiser F E, Perry H M. 3rd, Patrick P, Morley P M, Stauber P M, Vellas B, Baumgartner R N, Garry PJ (1997). Longitudinal changes in testosterone, luteinizing hormone, and follicle stimulating hormone in healthy older men. Metabolism; 46: 410-413. Nakagawa K, Obara T, Matsubara M, Kubo M (1982). Relationship o f changes in serum concentrations o f changes in serum concentrations o f prolactin and testosterone during dopaminergic modulation in males. J. Clin. Endocrinol; 17: 345-52. Nakashima A, Koshiyama K, Oozumi T (1975). Effect o f general anaesthesia and severity of surgical stress on serum LH and testosterone in males. Acta Endocrinol; 78:258-69. Nilson B, So'dergard R, Damber MG, Dambar JE, Von Schoultz B (1983). Free testosterone levels during Danazol therapy. Fertil Steril; 39: 55-9. 92 University of Ghana http://ugspace.ug.edu.gh Nocke-Finck L, Breuer H, Reimers D (1980). Wirkin von rifampicin and Streptomysin auf die Koszentration von Testosterone and Cortisol in Blutt von Mannem. J. Clin Chem Clin Biochem; 18: 897-9 (English Abstr). O ’Brien IA, Lewin IG, O’Hare JP, Corail RJM (1982). Reversible male subfertility due to hyperthyroidism. Br. Med J; 285:691 Olivo J, Southern AL, Gordon GG, Tochimoto S (1970). Studies o f the protein binding of testosterone in plasma in disorders o f thyroid function. Effect on therapy; J Clin Endocrinol. 31: 539-45 Oyama T, Kudo T (1972). Effect o f thiopentone, nitrous oxide anaesthesia and surgery on plasma testosterone levels in human males. Br J Anaes; 44: 704-6. Palmer RL, Crisp AH, Mackinnon PCB (1975). Pituitary sensitivity to 50|j.g LH/FSH-RH in subjects with anorexia nervosa in acute and recovery stages. Br Med J; 1: 179-82. Pang S, Levine LS, Chow E (1979). Di-hydrotestosterone and its relationship to testosterone in infancy and childhood. J Clin Endocrinol Metab; 49: 132-7. Pedan NR, Boyd EJS, Browning MCH, Saunder JHBB, Wormsley KG (1981). Effect of two-histamine H2 receptor blocking drugs on basal levels of gonadotrophins, prolactin, testosterone and oestradiol-17|3 during treatment of duodenal ulcer in male patients. Acta Endocrinol; 15 : 564-8. 93 University of Ghana http://ugspace.ug.edu.gh Peterson RE, Imperaqtor-McGenely J, Gautier T (1977). Male pseudo-hemaphroditism due to steroid 5a-reductase deficiency. Am J Med; 62:170-91. Pukkinen MO, Maepaa J (1983). Decrease in serum testosterone concentration during treatment with tetracycline. Acta Endocrinol. 103: 269-2. Ridgeway EC, Longscope C, Maloof F (1975). Metabolic clearance and blood production rates of oestradiol in hyperthyroidism J. Clin. Endocrinol Metab. 4: 491-7 Rilling JK, Wortham CM, Campbell BC, Stallings JF, Mbizva M (1996). Ratio o f plasma and testosterone throughout puberty: Production versus bioavailability. Steroid; 61/6: 374­ 378; Ross LS (1983). Diagnosis and treatment of infertile men. A clinical perspective. J Urol; 130: 8847-54 Ross R, Bernstein A, Zudd HET (1986). Serum Testosterone levels in healthy young black and white men. JNCL; 76:45-48 Rudd BT (1983). Urinary 17-Oxogenic and 17-oxosteroids. A case for detection from clinical chemistry repertoire. Ann Clin Biochem; 20: 65-71. Ruder H, Coriol P, Mahoudeau JA, Ross GT, Lipsett MB (1971). Effects of induced hyperthyroidism on steroid metabolism in man. J Clin Endocrinol Metab; 33:382-7 94 University of Ghana http://ugspace.ug.edu.gh Rudman D, Feller AG, Nagrey HS, Gergans GA, Lalitha PV, Goldberg AF, Schlenker, RA, Coh, L, Rudman W, Mattson DE (1990). Effects o f human growth hormone in men over 60 years old. N Engl J Med; 323: 16 Saenger P, Goldman AS, Livine LS (1978). Prepubertal diagnosis o f steroid 5a- reductase deficiency. J Clin Endocrinol Metab; 46:627-34. Saenger P (1979). Steroid 5a reductase deficiency. In: The intersex child Paediatr Adolesce Endocrinol; Vol. 8. Basel: Karger, 156-70. Saenger P (1984). Abnormal sex differentiation. J Paediatr; 104: 1-17. Semple PD’a, Beastall GH, Watson WS (1980). Serum testosterone depression associated with hypoxia in respiratory failure. Clin Sci; 58:106-6. Savage MO (1982). Ambiguous genitalia, small genitalia and un-descended testis. Clin Endocrinol Metab; 11: 127-58. Savarino V, Giusti M, Mansi C, Marugo M, Delitate G, Celle G (1983). Ranitidine and testosterone secretion in man. Br J Clin Pharmacol; 15 : 579. Schulster A, Ross L, Scommegna A (1983). Frequency of androgen insensitivity in infertile phenotypicapically normal men. J Urol; 130: 699-701. 95 University of Ghana http://ugspace.ug.edu.gh Schwartz M, Imperato-McGinley J, Peterson RE (1981). Male pseudo-hermaphroditism secondary to an abnormality in Leydig cell differentiation. J Clin Endocrinol Metab; 53: 123-7. Schwarz S, Boyd J (1982). Interference o f Danazol with the radioimmunoassay o f steroid hormones. JSteroid Biochem; 16: 823-6. Siiteri PK, Wilson JD (1974). Testosterone formation and metabolism during male sexual differentiation on the human embryo. J Clin Endocrinol Metab; 38: 113-25. Siris S, Siris ES, Van Kammen DP (1980). Effects o f dopamine blockade on gonadotrophin and testosterone in men. Amer J Psychiatr; 137:211-4. Spandri P, Gangemi M, Nardelli GB (1984). Testosterone, 17KS, Oestradiol, FSH-LH variation and hirsutism modification during spironolactone therapy. Clin Exp. Obstet Gynaecol; 11:49-54. Spark R., F., White R.,A., Connoly P.,B. (1980). Impotency in not always psychogenic JAMA; 243: 750-5. Spitz L (1983). Maldescent o f the testis. Arch Dis Child; 59:847-8. Synder PJ, Peachey H, Hannoush P, Berlin JA, Loh L, Holmes JA, Dlewati A, Staley J, Santanna J, Kapoor SC, Altie MF, Haddad JG Jr, Strom, B.L. (1999 a) Effect of testosterone treatment on body composition and Muscle strength in men over 65 years of age. J Clin Endocrinol Metab; 84:2647-2653. University of Ghana http://ugspace.ug.edu.gh Synder PT, Peachey H, Hannoush P, Berlin JA, Loh L, Holmes JA, Dlewati A, Staley.J, Santanna, J, Kapoor SC, Altie M.F, Haddad, JG Jr, Strom BL (1999b). Treatment on bone mineral density in men over 65 years of age. J Clin Endocrinol Metab; 84: 1966-1972. Tapanainen J (1983). Hormonal changes during the perinatal period: Serum testosterone; some o f its precursors and FSH and prolactin in pre-term and full term male infants, cord blood and during first week of life. J Steroid Biochem; 18: 13-18. Tibblin G, Adlerberth A, Liaclstedt G, Bjomtorp P (1996). The pituitary gonadal axis and health in elderly men: a study o f men bom in 1913. Diabetes; 45 (11): 1606-9 Tietz N.W. (1990). Clinical Guide to Laboratory Tests; Second Edition. Edited by Norbert W. Tietz. W.B. Saunders Company. Philadelphia, London, Toronto, Montreal, Sydney, Tokyo. Toone BK, Wheeler M, Nanjee M. (1982). Sex hormones sexual activity and plasma anticonvulsant levels in male epileptics. N. J Neurol Neurone Psychiatr; 46: 824-6. Toone BK, Wheeler M, Nanjee M, Fenwick PBC (1980). Sex hormone changes in male epileptics. Clin Endocrinol; 12: 391-5 Totowa Meikle A.W.(1999). Androgen replacement therapy and male hypogonadism. In contemporary Endocrinology: Hormone Replacement Therapy (ed. A.W. Meikle). Pp 263­ 306. Humana Press Inc; Totowa. 97 University of Ghana http://ugspace.ug.edu.gh Uygur MC, Arik Al, Altug U, Erol D (1998). Effects of the 5 alpha reductase inhibitor finasteride on serum levels of gonadal, adrenal, and hypophyseal hormones and its clinical significance: a prospective clinical study. Steroid. 63 (4): 208-13 Valero-Politie J, Fuentes Arder X (1996). Daily rhythmic and non-rhythmic variations of follitropin, lutropin, testosterone and sex hormone binding globulin in men. Euro J Clin Chem and Clin Biochem; 34(6): 455-62 Van Kammen E, Thijssen JHH, Schwartx F (1978). Sex hormones in male patients with chronic renal failure. Clin Endocrinol; 8:7-14. Van Thiel DH, Lester R (1979). The effects of chronic alcohol abuse on sexual function. Clin Endorinol Metab; 8: 499-510. Vermeulen A Kaufman JM, Giagulli VA (1996). The influence o f some biological indexes on sex hormone binding globulin and androgen levels in aging or obese males. J Clinical Endocrinol Metab; 81 (5): 1821-6. Vermeulen A, (1984). Hirsutism and virilism. Med Int; 12: 501-5. Wang C, Lai CL, Lai XC, Yeung KK (1982). Effect o f cimetidine on gonadal function in man. Br J Clin Pharmacol; 13:791-4. 98 University of Ghana http://ugspace.ug.edu.gh Wang C, Iranmanesh A, Berman N, McDonald V, Steiner B, Ziel F, Faukner SM, Dudley RE, Veldhuis JD, SwerdloffRS (1998). Comparative pharmaco-kinetics o f three doses of percutaneous di-hydroxytestosterone gel in healthy elderly men, a clinical research centre studies. J Clin Endocrinol Metab; 83 (8): 2479-57. Whitby LG, Smith AF, Beckett GJ, Walker SW (1993). Lecture notes on Clinical Biochemistry. 5th Edition pp 304. Oxford. Blackwell Scientific Publications. Whitby RM, Gordon RD, Blair BR (1979). The endocrine effects o f vasectomy: A prospective five year study. Fertil Steril; 31: 518-20. Wilson JD, Griffin JE, George FW (1981). The role o f gonadal steroids in sexual differentiation. Rec Progr Horm Res; 37:1-33 Winter JSD,Tarska S, Faiman (1972). The hormonal response to HCG stimulation in male children and adolescents. J Clin Endocrinol Metab; 34:348-353. Winter JSD (1982). Hypothalamic-pituitary function in the fetus and infant. Clin.Endocrinol Metab; 11: 41-45. Winter SJ, Atkinson L (1997). Serum LH concentrations in hypogonadal men during transdermal testosterone replacement through scrotal skin, further evidence that ageing enhances testosterone negative feedback. The Testoderm Study Group. Clin Endocrinol. 47 (3): 317-22. 99 University of Ghana http://ugspace.ug.edu.gh Winter SJ, Brufsky A, Weisfield J, Trump DL, Dyky MA, Hadeed V (2001). Metabolism; 50 (10): 1242 -7 Wu FC, Balasubramanian R, Mulders TM, Coaelingh-Bennink HJ (1999). Oral progestogen combined with testosterone as a potential male contraceptive; additive effects between desogestral and testosterone ethanthate in suppression o f spermatogenesis, pituitary testicular axis and lipid metabolism. J Clin Endocrinol Metab. 84 (1): 112-22 Wu FCQ, Edmond P, Raab G (1981). Endocrine assessment o f the subfertile male. Clin Endocrinol; 14: 493-507. Yen SSC (1980). The polycystic ovarian syndrome. Clin Endocrinol .12:177-208. Zmuda JM, Thompson PD, Winters SJ (1996). Exercise increases serum testosterone and sex Hormone binding globulin in older men. Metabolism Clinical & Experimental. 45 (8):35-9. Zumoff B, Strain GW, Miller Lk (1990) Plasma free and non-sex-hormone- binding-globulin bound testestertone are decreased in obese men in proportion to their degree of obesity. J Clin Endocrinol Metab ; 71: 929-31. 100 University of Ghana http://ugspace.ug.edu.gh APPF.NniY i QUESTION AIRE FOR TF.STO STERONE 1 • Number:............................................................................................... 2. Name:........................................................................Age/Sex:......... 3. Weight:................................................. Height:................................. 4. Location:............................................................................................. 5. Address:............................................................................................. 6. Marital Status:.............................. Single:.........................Married: 7. Drug History:..................................................................................... 8. Past History of Illness:..................................................................... 9. No. o f Living Children: 10. Eating Habit:................ 101 University of Ghana http://ugspace.ug.edu.gh APPENDIX II n a m e AGE TESTO. SHBG LH FSH J.M.M 34 29.37 17.61 4.0 5.4 H.Y.O 35 30.21 51.34 3.4 6.0 K.B 34 24.49 48.31 10.2 9.5 M.C. 42 18.58 32.86 6.6 5.4 W.C.M. 57 28.72 47.50 14.9 7.8 P.H. 22 33.56 52.96 7.9 4.5 L.D 23 37.98 59.92 7.3 8.0 E.A.Y 28 28.77 58.31 9.0 6.2 R.S 23 32.40 53.76 5.6 6.7 T.B.W 22 36.40 48.82 15.7 64.5 G.S.S. 67 22.91 32.45 8.6 6.89 C.A 23 32.95 31.34 3.4 8.6 S.A.M 32 36.82 72.75 5.9 4.9 A.Q 46 36.35 50.94 7.5 7.5 102 University of Ghana http://ugspace.ug.edu.gh a .a .h 47 29.56 30.93 16.7 18.3 D.K 37 31.75 19.82 16.0 20.0 P.A 52 34.21 17.60 16.0 10.5 A.S 42 36.47 40.83 6.5 7.5 N.M.A 40 26.03 14.07 13.3 6.8 S.M 35 26.79 28.91 6.8 5.5 K.P.A.M 42 28.214 17.40 4.1 3.5 B.A.A. 40 32.68 24.27 10.4 7.2 M.K.W. 41 38.77 64.37 6.1 4.1 W.A. 52 29.79 42.96 4.0 7.3 N.G. 45 35.10 29.82 6.0 4.2 E.G 23 37.52 43.66 11.1 10.5 J.A. 42 37.10 83.66 11.8 7.1 P.T 37 41.75 88.61 4.5 4.1 S.A 32 30.82 40.43 4.2 2.4 A.B 21 39.19 45.18 9.2 3.3 l.O.N. 39 34.17 32.35 7.0 5.3 103 University of Ghana http://ugspace.ug.edu.gh — — _ E.A 23 36.35 41.35 15.3 8.9 G-A.M 54 40.35 70.33 5.3 2.7 G.O. 25 35.52 36.09 6.7 7.0 S.K.A. 54 34.03 52.25 6.3 9.5 J.A.A. 42 40.21 75.28 15.2 9.5 F.O.K 47 30.52 33.97 11.6 13.2 D.A. 46 32.21 20.23 4.8 3.6 S.A. 32 36.07 22.25 5.1 8.1 J.B.A. 49 31.56 51.04 11.8 9.2 J.A. 53 34.40 38.81 30.6 38.5 R.B.A. 45 31.42 28.21 8.3 3.9 C.S 36 32.26 23.26 82.0 28.5 EA . 51 29.47 22.75 8.5 7.,7 O.D. 63 34.40 73.56 2.7 5.9 A.C. 51 30.12 40.03 12.8 7.3 L.T.O.Q. 61 30.3 24.77 9.7 12.7 G A . 68 38.82 75.38 5.3 4.6 104 University of Ghana http://ugspace.ug.edu.gh D.D. 47 37.10 31.94 8.6 6.0 k .b . 58 35.47 46.29 2.3 4.5 e .a .a . 41 24.68 13.36 51.6 40.5 R.B. 54 27.66 27.60 5.4 6.7 D.C 61 28.57 17.10 3.7 2.4 T.A. 39 37.66 47.50 4.0 4.8 O.S 33 33.10 42.75 13.4 2.9 A.R.S. 24 33.10 18.01 3.6 2.4 J O 28 32.03 15.18 10.7 1.9 E.B 41 35.47 35.38 12.8 32.8 E.B. 15 6.03 71.14 8.4 5.4 E.S. 30 38.17 36.18 8.0 9.1 E.A. 28 35.10 27.70 3.,9 3.1 N.D.D. 24 36.35 19.22 7.4 8.4 P.A. 59 35.98 9.66 6.9 8.7 S.L 42 36.21 35.99 12.8 6.9 A.D 14 31.24 12.75 9.1 5.0 105 University of Ghana http://ugspace.ug.edu.gh Y.P 17 32.86 23.26 2.6 3.4 Q.S 14 0.44 45.18 3.2 2.6 J.c. 16 28.63 27.70 2.4 5.4 G.C 16 5.27 60.13 3.0 10.3 S.A 11 5.6 91.24 2.6 1.5 i.e . 12 4.76 82.05 3.2 2.5 A.L 15 15.24 49.22 3.2 7.6 B.L 17 24.91 12.45 4.8 5.6 I.Y 18 38.95 44.96 2.8 5.3 A.B.A 18 29.79 39.92 76.3 12.8 L.O 61 23.05 12.96 20.6 6.6 E.G 57 29.70 60.03 2.0 6.0 N.D.D. 52 29.84 29.92 4.7 2.8 T.K.K. 25 36.72 30.23 2.4 5.0 E.N 15 11.55 32.15 3.1 2.7 D.A. 13 7.79 16.79 38.1 2.5 K.O.W 67 24.03 45.78 4.6 6.7 106 University of Ghana http://ugspace.ug.edu.gh E.A. 58 58.16 40.13 6.9 3.8 T.O.A. 56 56.54 15.18 0.5 4.1 EIA Control 1 1.2 34.2 EIA Control 2 8.9 - - Randox 1 3.9 - 0.88 1.03 Control Randox 2 16.2 65.4 110 Control Cutoff point for testosterone analysis was 50.0nmol/L. Cutoff point for SHBG analysis was 70.0nmol/L. Cutoff point for LH analysis was 30.0U/L. Cutoff point for FSH analysis was 25.0U/L. 107 University of Ghana http://ugspace.ug.edu.gh A PPE N D IX III Comparison o f one EIA kit with two RIA kits. DPC (RIA) Fenzia (EIA) Farm Ext. (RIA) Controls Values. 2.10 T 185 2.4 1.57 2.37 1.80 T187 1.5 0.88 1.59 2.00 T192 2.3 1.78 2.21 18.55 T159 16.0 15.73 13.39 10.80 T165 11.2 10.59 10.47 31.90 T181 25.3 25.4 22.71 d reasonab ly with the control From the results it could be seen that the EIA results comparei values. 108